Anti-vegf protein compositions and methods for producing the same

ABSTRACT

The present disclosure pertains to compositions comprising anti-VEGF proteins and methods for producing such compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/211,333, filed on Mar. 24, 2021, which is a continuation of U.S.patent application Ser. No. 16/996,042, filed on Aug. 18, 2020, whichclaims priority to and the benefit of U.S. Provisional PatentApplication No. 63/065,012, filed on Aug. 13, 2020, the content of whichis incorporated herein by reference in its entirety. This applicationalso claims priority to and the benefit of Provisional PatentApplication No. 62/944,635, filed on Dec. 6, 2019.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on May 16, 2022, is named SL(05-16-22). txt and is 148,907 bytes in size.

FIELD

The present invention generally pertains to anti-VEGF compositions andmethods for producing the same.

BACKGROUND

Protein-based biopharmaceutical compositions have emerged as importantproducts for research, the treatment of ophthalmological diseases,cancer, autoimmune disease, and infection, as well as other diseases anddisorders. Biopharmaceuticals represent one of the fastest growingproduct segments of the pharmaceutical industry.

A class of cell-derived dimeric mitogens with selectivity for vascularendothelial cells has been identified and designated vascularendothelial cell growth factor (VEGF).

Persistent angiogenesis may cause or exacerbate certain diseases such aspsoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabeticretinopathy and neovascular glaucoma. An inhibitor of VEGF activitywould be useful as a treatment for such diseases and other VEGF-inducedpathological angiogenesis and vascular permeability conditions, such astumor vascularization. The angiopoietins and members of the vascularendothelial growth factor (VEGF) family are the only growth factorsthought to be largely specific for vascular endothelial cells.

Several eye disorders are associated with pathological angiogenesis. Forexample, the development of age-related macular degeneration (AMD) isassociated with a process called choroidal neovascularization (CNV).Leakage from the CNV causes macular edema and collection of fluidbeneath the macula resulting in vision loss. Diabetic macular edema(DME) is another eye disorder with an angiogenic component. DME is themost prevalent cause of moderate vision loss in patients with diabetesand is a common complication of diabetic retinopathy, a diseaseaffecting the blood vessels of the retina. Clinically significant DMEoccurs when fluid leaks into the center of the macula, thelight-sensitive part of the retina responsible for sharp, direct vision.Fluid in the macula can cause severe vision loss or blindness.

Various VEGF inhibitors, such as the VEGF trap EYLEA® (aflibercept),have been approved to treat such eye disorders.

SUMMARY

The present invention relates to anti-VEGF proteins including the VEGFtrap protein aflibercept, which is a fusion protein. The instantinvention also pertains to a new anti-VEGF protein, the afliberceptMiniTrap or VEGF MiniTrap (collectively referred to as MiniTrap unlessotherwise noted). Disclosed herein are methods of making these anti-VEGFproteins, including production modalities that provide efficient andeffective means to produce the proteins of interest. In one aspect, theinstant invention is directed towards the use of chemically definedmedia (CDM) to produce anti-VEGF proteins. In a particular aspect, theCDMs of interest are those that, when used, produce a protein samplewherein the sample has a yellow-brown color and may comprise oxidizedspecies. Still further in the present application, protein variants ofaflibercept and VEGF MiniTrap are disclosed together with attendantproduction methods.

Production of Aflibercept

The present disclosure describes the production of aflibercept using acell culture medium. In one embodiment, the cell culture medium is achemically defined medium (“CDM”). CDM is often used because it is aprotein-free, chemically-defined formula using no animal-derivedcomponents and there is certainty as to the composition of the medium.In another embodiment, the cell culture medium is a soy hydrolysatemedium.

In one embodiment, a method of producing a recombinant proteincomprises: (a) providing a host cell genetically engineered to express arecombinant protein of interest; (b) culturing the host cell in a CDMunder suitable conditions in which the cell expresses the recombinantprotein of interest; and (c) harvesting a preparation of the recombinantprotein of interest produced by the cell. In one aspect, the recombinantprotein of interest is an anti-VEGF protein. In a particular aspect, theanti-VEGF protein is selected from the group consisting of afliberceptand recombinant MiniTrap (examples of which are disclosed in U.S. Pat.No. 7,279,159), an aflibercept scFv and other anti-VEGF proteins. In apreferred aspect, the recombinant protein of interest is aflibercept.

In one aspect of the present embodiment, aflibercept is expressed in asuitable host cell. Non-limiting examples of such host cells include,but are not limited to, CHO, CHO K1, EESYR®, NICE®, NSO, Sp2/0,embryonic kidney cells and BHK.

Suitable CDMs include Dulbecco's Modified Eagle's (DME) medium, Ham'sNutrient Mixture, Excell medium, and IS CHO-CD medium. Other CDMs knownto those skilled in the art are also contemplated to be within the scopeof the present invention. In a particular aspect, a suitable CDM isCDM1B (Regeneron) or Excell Advanced Medium (SAFC).

In one embodiment, a clarified harvest sample from a CDM culturecomprising aflibercept is subjected to a capture chromatographyprocedure. In one aspect, the capture step is an affinity chromatographyprocedure using, for example, Protein A. In a further aspect, the eluateof the affinity procedure exhibits a certain color, for example, theeluate can exhibit a yellow-brown color. As described in more detailinfra, color can be assessed using (i) the European Color Standard “BY”in which a qualitative visual inspection is made or (ii) a colorimetricassay, CIE L*, a*, b* (or CIELAB), which is more quantitative than theBY system. However, in either case, color assessment between multiplesamples should be normalized against protein concentration in order toassure a meaningful assessment. For example, referring to Example 9below, the Protein A eluate has a “b*” value of around 2.52 whichcorresponds to a BY value of approximately BY5 (when measured at aconcentration of 5 g/L protein in the protein A eluate). If the color ofthe Protein A eluate is to be compared to another sample, then thecomparison should be made against the same protein concentration. The b*value in the CIELAB color space is used to expresses coloration of thesamples and covers blue (−) to yellow (+). A higher b* value of a samplecompared to another indicates a more intense yellow-brown coloration inthe sample compared to the other.

In one embodiment, aflibercept is produced from a host cell geneticallyengineered to express aflibercept using CDM. In one aspect, otherspecies or variants of aflibercept are also produced. These variantsinclude aflibercept isoforms that comprise one or more oxidized aminoacid residues collectively referred to as oxo-variants. A clarifiedharvest sample produced using CDM comprising aflibercept as well as itsoxo-variants can be subjected to a capture chromatography procedure. Inone aspect, the capture step is an affinity chromatography procedureusing, for example, a Protein A column. When a sample extracted from anaffinity eluate, which may or may not manifest a yellow-brown color, isanalyzed using, for example, liquid chromatography-mass spectrometry(LC-MS), one or more oxidized variants of aflibercept may be detected.Certain amino acid residues of a modified aflibercept are shown to beoxidized including, but not limited to, histidine and/or tryptophanresidues. In one aspect, the variants can include oxidation of one ormore methionine residues as well as other residues, see infra.

In another aspect, the variants can include oxidation of one or moretryptophan residues to form N-formylkynurenine. In a further aspect, thevariants can include oxidation of one or more tryptophan residues toform mono-hydroxyl tryptophan. In a particular aspect, the proteinvariants can include oxidation of one or more tryptophan residues toform di-hydroxyl tryptophan. In a particular aspect, the proteinvariants can include oxidation of one or more tryptophan residues toform tri-hydroxyl tryptophan.

In another aspect, the variants can include one or more modificationsselected from the group consisting of: deamidation of, for example, oneor more asparagines; one or more aspartic acids converted toiso-aspartate and/or Asn; oxidation of one or more methionines;oxidation of one or more tryptophans to N-formylkynurenine; oxidation ofone or more tryptophans to mono-hydroxyl tryptophan; oxidation of one ormore tryptophans to di-hydroxyl tryptophan; oxidation of one or moretryptophans to tri-hydroxyl tryptophan; Arg 3-deoxyglucosonation of oneor more arginines; removal of C-terminal glycine; and presence of one ormore non-glycosylated glycosites.

In another embodiment, the invention is directed to methods forproducing aflibercept. In one aspect, a clarified harvest samplecomprising aflibercept and its variants are subjected to a capture stepsuch as Protein A affinity chromatography. Subsequent to the affinitystep, an affinity eluate can be subjected to ion exchangechromatography. The ion exchange chromatography can be either cation oranion exchange chromatography. Also contemplated to be within the scopeof the present embodiment is mixed-mode or multimodal chromatography aswell as other chromatographic procedures which are discussed furtherbelow. In a particular aspect, the ion exchange chromatography is anionexchange chromatography (AEX). Suitable conditions for employing AEXinclude, but are not limited to, Tris hydrochloride at a pH of about 8.3to about 8.6. Following equilibration using, for example, Trishydrochloride at a pH of about 8.3 to about 8.6, the AEX column isloaded with sample. Following the loading of the column, the column canbe washed one or multiple times using, for example, the equilibratingbuffer. In a particular aspect, the conditions used can facilitate thedifferential chromatographic behavior of aflibercept and its oxidizedvariants such that a fraction comprising aflibercept absent significantamounts of oxo-variants can be collected in a flowthrough fraction whilea significant portion of oxo-variants are retained on the solid-phase ofthe AEX column and can be obtained upon stripping the column—see Example2 below, FIG. 11. Referring to FIG. 11 and Example 2, changes inoxo-variants can be observed between the different production steps. Forexample, this change can be illustrated by data in the “TryptophanOxidation Level (%)” section, specifically, the “W138(+16)” column.There it can be observed that the oxo-variants (specifically,oxo-tryptophan) went from about 0.131% in a load sample to about 0.070%in a flowthrough sample following AEX chromatography (AEX separation 2),indicating that there was a reduction in oxo-variants of afliberceptusing AEX.

Use of ion exchange can be used to mitigate or minimize color. In oneaspect of the present embodiment, a clarified harvest sample issubjected to capture chromatography, for example, using Protein Aaffinity chromatography. The affinity column is eluted and has a firstcolor with a particular BY and/or b* value assigned thereto. ThisProtein A eluate is then subjected to ion exchange chromatography suchas anion exchange chromatography (AEX). The ion exchange column iswashed and the flowthrough is collected and has a second color having aparticular BY and/or b* value assigned thereto. In a particular aspect,the color value (either “BY” or “b*”) of the first color differs fromthe second color. In a further aspect, the first color of the Protein Aeluate has a more yellow-brown color as compared to the second color ofthe AEX flowthrough as reflected by the respective BY and/or b* value.Typically, there is a reduction in yellow-brown color of the secondcolor following AEX when compared to the first color of the Protein Aeluate. For example, the use of anion exchange reduced the yellow-browncolor observed in a Protein A eluate sample from a b* value of about3.06 (first color) to about 0.96 (second color) following AEX—seeExample 2, Table 2-3 below.

In one aspect of the embodiment, the pH of both the equilibration andwash buffers for the AEX column can be from about 8.30 to about 8.60. Inanother aspect, the conductivity of both the equilibration and washbuffers for the AEX column can be from about 1.50 to about 3.00 mS/cm.

In one aspect of the embodiment, the equilibration and wash buffers canbe about 50 mM Tris hydrochloride. In one aspect, the strip buffercomprises 2 M sodium chloride or 1 N sodium hydroxide or both (see Table2-2).

The present embodiment can include the addition of one or more steps, inno particular order, such as hydrophobic interaction chromatography(HIC), affinity chromatography, multimodal chromatography, viralinactivation (e.g., using low pH), viral filtration, and/orultra/diafiltration as well as other well-known chromatographic steps.

In one embodiment, the anti-VEGF protein is glycosylated at one or moreasparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAcglycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation;G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation;G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3glycosylation; G0-2G1cNAc glycosylation; Man4 glycosylation; Man4_A1G1glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1glycosylation; Man5 A1G1S1 glycosylation; Man6 glycosylation;Man6_G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/orMan7 glycosylation. In one aspect, the anti-VEGF protein can beaflibercept, anti-VEGF antibody or VEGF MiniTrap.

In one aspect, glycosylation profile of a composition of an anti-VEGFprotein is as follows: about 40% to about 50% total fucosylated glycans,about 30% to about 55% total sialylated glycans, about 6% to about 15%mannose-5, and about 60% to about 79% galactosylated glycans (seeExample 6). In one aspect, the anti-VEGF protein has Man5 glycosylationat about 32.4% of asparagine 123 residues and/or about 27.1% ofasparagine 196 residues.

In one embodiment, the process can further comprise formulating a drugsubstance using a pharmaceutically acceptable excipient. In one aspectof the embodiment, the pharmaceutically acceptable excipient can beselected from the following: water, buffering agents, sugar, salt,surfactant, amino acid, polyol, chelating agent, emulsifier andpreservative. Other well-known excipients to the skilled artisan arewithin the purview of this embodiment.

In one aspect of the embodiment, the formulation can be suitable foradministration to a human subject. In particular, administration can beaffected by intravitreal injection. In one aspect, the formulation canhave about 40 to about 200 mg/mL of the protein of interest.

The formulation can be used as a method of treating or preventingangiogenic eye disorders which can include: age-related maculardegeneration (e.g., wet or dry), macular edema, macular edema followingretinal vein occlusion, retinal vein occlusion (RVO), central retinalvein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabeticmacular edema (DME), choroidal neovascularization (CNV), irisneovascularization, neovascular glaucoma, post-surgical fibrosis inglaucoma, proliferative vitreoretinopathy (PVR), optic discneovascularization, corneal neovascularization, retinalneovascularization, vitreal neovascularization, pannus, pterygium,vascular retinopathy, diabetic retinopathy in a subject with diabeticmacular edema; or diabetic retinopathies (e.g., non-proliferativediabetic retinopathy (e.g., characterized by a Diabetic RetinopathySeverity Scale (DRSS) level of about 47 or 53) or proliferative diabeticretinopathy; e.g., in a subject that does not suffer from DME).

Production of VEGF MiniTrap

The present disclosure describes the production of a modified version ofaflibercept wherein the Fc portion is removed or absent and is referredto as aflibercept MiniTrap or VEGF MiniTrap. This MiniTrap can beproduced in cell culture medium including a chemically defined medium(CDM) or soy hydrolysate medium.

In one embodiment, the MiniTrap is produced using CDM. In one aspect ofMiniTrap production, full length aflibercept is produced using asuitable host and under suitable conditions and is further processedwhereby the Fc portion is enzymatically removed thus yielding MiniTrap.Alternatively, a gene encoding MiniTrap (e.g., a nucleotide sequenceencoding aflibercept absent its Fc portion) can be produced undersuitable conditions using a suitable host cell.

In one embodiment, a method for manufacturing MiniTrap includesproducing a full-length aflibercept fusion protein followed by cleavageof the Fc region. In one aspect, the method involves producing arecombinant protein, namely a full-length aflibercept fusion protein(see, U.S. Pat. No. 7,279,159, the entire teaching of which isincorporated herein by reference), comprising: (a) providing a host cellgenetically engineered to express full length aflibercept; (b) culturingthe host cell in CDM under suitable conditions in which the cellexpresses the full length aflibercept; (c) harvesting a preparation ofthe full length aflibercept produced by the cell; and (d) subjecting thefull length aflibercept to enzymatic cleavage specific for removing theFc portion of the fusion protein. In another aspect, a nucleotidesequence encoding aflibercept minus its Fc portion is expressed from asuitable host cell under suitable conditions well known to those skilledin the art (see U.S. Pat. No. 7,279,159).

In one aspect of the present embodiment, the aflibercept is expressed ina suitable host cell. Non-limiting examples of such host cells include,but are not limited to, CHO, CHO K1, EESYR®, NICE®, NSO, Sp2/0,embryonic kidney cells and BHK.

Suitable CDMs include Dulbecco's Modified Eagle's (DME) medium, Ham'sNutrient Mixture, EX-CELL medium (SAFC), and IS CHO-CD medium (Irvine).Other CDMs known to those skilled in the art are also contemplated to bewithin the scope of the present invention. In a particular aspect, asuitable CDM is CDM1B (Regeneron) or Excell medium (SAFC).

In one aspect, during the production of MiniTrap, a sample comprising aprotein of interest (i.e., aflibercept fusion protein and/or MiniTrap)along with its variants (including oxo-variants) can exhibit certaincolor properties—a yellow-brown color. For example, an eluate samplefrom an affinity chromatography step can exhibit a certain yellow-browncolor measured using the BY and/or b* system (see Examples 2 and 9below). Exemplary sources for a “sample” may include an affinitychromatography, such as Protein A, eluate; the sample may be obtainedfrom a flowthrough fraction of ion exchange chromatography procedure; itmay also be obtained from the strip of an ion exchange column—there areother sources during a production process well known to those skilled inthe art from which a sample may be analyzed. As mentioned above anddescribed further below, color can be assessed using (i) the EuropeanColor Standard “BY” in which a qualitative visual inspection is made or(ii) a colorimetric assay, CIELAB, which is more quantitative than theBY system. However, in either case, color assessment between multiplesamples should be normalized, for example, using protein concentration,in order to assure a meaningful assessment between samples.

In one aspect of the present embodiment, a full-length afliberceptfusion protein can be subjected to enzymatic processing (“cleavageactivity”) in order to generate a VEGF MiniTrap, for example, usingproteolytic digestion employing a protease or enzymatically activevariant thereof. In one aspect of this embodiment, the protease can bean immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS). Inanother aspect, the protease can be thrombin trypsin, endoproteinaseArg-C, endoproteinase Asp-N, endoproteinase Glu-C, outer membraneprotease T (OmpT), IdeS, chymotrypsin, pepsin, thermolysin, papain,pronase, or protease from Aspergillus saitoi. In one aspect, theprotease can be a cysteine protease. In a particular aspect of theembodiment, the protease can be IdeS. In another aspect, the proteasecan be a variant of IdeS. Non-limiting examples of variants of IdeS aredescribed infra and include a polypeptide having an amino acid sequenceas set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3,SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.:8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ IDNO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In oneaspect, the protease can be immobilized on agarose or another suitablematrix.

In one aspect, a protein of interest (together with its variants) isproduced using CDM. In a particular aspect, the protein of interestincludes aflibercept or MiniTrap. The variants comprise one or moreoxidized amino acid residues, collectively oxo-variants. Examples ofoxidized residues include, but are not limited to, one or more histidineand/or tryptophan residues. Other oxidized residues have also beendetected using LC-MS and are described below, such as oxidizedmethionine. Subsequent chromatography such as AEX can be used to isolatethese oxo-variants from the protein of interest in a given sample andare described herein.

In one aspect, the variants can include oxidation of one or moretryptophan residues to form N-formylkynurenines. In a further aspect,the variants can include oxidation of one or more tryptophan residues toform mono-hydroxyl tryptophan. In a particular aspect, the proteinvariants can include oxidation of one or more tryptophan residues toform di-hydroxyl tryptophan. In a particular aspect, the proteinvariants can include oxidation of one or more tryptophan residues toform tri-hydroxyl tryptophan.

In another aspect, the oxo-variants can include one or moremodifications selected from the group consisting of: deamidation of oneor more asparagine residues; one or more aspartic acids converted toiso-aspartate and/or asparagine; oxidation of one or more methionineresidues; oxidation of one or more tryptophan residues to formN-formylkynurenine; oxidation of one or more tryptophan residues to formmono-hydroxyl tryptophan; oxidation of one or more tryptophan residuesto form di-hydroxyl tryptophan; oxidation of one or more tryptophanresidues to form tri-hydroxyl tryptophan; Arg 3-deoxyglucosonation ofone or more arginine residues; removal of C-terminal glycine; andpresence of one or more non-glycosylated glycosites.

In one embodiment, the method of manufacturing a MiniTrap proteincomprises (a) capturing a full-length aflibercept fusion protein on afirst chromatographic platform and (b) cleaving the aflibercept therebyforming a MiniTrap protein, i.e., aflibercept absent its Fc domain. Inone aspect, the first chromatographic support comprises an affinitychromatography media, an ion-exchange chromatography media, or ahydrophobic interaction chromatography media. In a particular aspect,the first chromatographic platform comprises an affinity chromatographyplatform such as a Protein A. In a further aspect, the protein ofcapture step (a) is eluted from the first chromatography platform priorto cleavage step (b). In a still further aspect, a second capture stepfollows cleavage step (b). In a particular aspect, this second capturestep can be facilitated by affinity chromatography such as Protein Aaffinity chromatography. The flowthrough of this second capture step(comprising MiniTrap) has a first color, for example, a yellow-browncolor and measured having a particular BY and/or b* value—see, e.g.,Example 9 below. Additionally, LC-MS analysis of this second captureflowthrough may demonstrate the presence of oxo-variants wherein one ormore residues of MiniTrap are oxidized (see Example 9 below).

In a further aspect, the second capture flowthrough can be subjected toion exchange chromatography such as AEX. This AEX column can be washedusing a suitable buffer and an AEX flowthrough fraction can be collectedcomprising essentially MiniTrap. This AEX flowthrough fraction can havea second color that is of a yellow-brown coloration having a particularBY and/or b* value. In a further aspect, the first color (flowthroughfrom second capture step) and second color (flowthrough of the ionexchange procedure) have different colors as measured either by the BYand/or b* system. In one aspect, the second color demonstrates adiminished yellow-brown color when compared to the first color usingeither a BY and/or b* value following AEX.

In another embodiment, the cleavage activity of step (b) can beperformed using a chromatographic column wherein the cleavage activity,for example, an enzyme activity, is affixed or immobilized to a columnmatrix. The column used in step (b) can comprise one or more of theproteases already alluded to and more fully described below.

In one embodiment, the ion-exchange chromatography procedure cancomprise an anion-exchange (AEX) chromatography media. In anotheraspect, the ion-exchange chromatography media comprises a cationexchange (CEX) chromatography media. Suitable conditions for employingAEX include, but are not limited to, Tris hydrochloride at a pH of about8.3 to about 8.6. Following equilibration using, for example, Trishydrochloride at a pH of about 8.3 to about 8.6, the AEX column isloaded with sample. Following the loading of the column, the column canbe washed one or multiple times using, for example, the equilibratingbuffer. In a particular aspect, the conditions used can facilitate thedifferential chromatographic behavior of MiniTrap and its oxo-variantsusing AEX such that the MiniTrap is substantially in the flowthroughfraction while the oxo-variants are substantially retained on the AEXcolumn and can be collected by stripping the column (see Example 9below).

In one example, samples from different stages of production wereanalyzed for color and presence of oxo-variants. Referring to Example 9,the affinity flowthrough pool (flowthrough from a second Protein Aaffinity step) had a first b* value of about 1.58 (see Table 9-3). Thissecond affinity flowthrough was subjected to AEX. The AEX flowthroughhad a second b* value of about 0.50, indicating a significant reductionin yellow-brown color following the use of AEX. Stripping of the AEXcolumn yielded a strip sample and a third b* value of about 6.10 wasobserved, indicating that this strip sample had a more yellow-browncolor when compared to either the load or flowthrough.

Referring again to Example 9, oxo-variant analysis was also performed.Samples analyzed were the affinity flowthrough pool (second Protein Aaffinity eluate), AEX flowthrough, and AEX strip. Referring to Table 9-5and Table 9-6, changes in oxo-variants can be observed between thedifferent production steps. For example, this change can be illustratedby data in the “Tryptophan Oxidation Level (%)” section, specifically,the “W58(+16)” column. There it can be observed that the oxo-variants(specifically, oxo-tryptophan) went from about 0.055% in a load sampleto about 0.038% in a flowthrough sample following AEX chromatography,indicating that there was a reduction in oxo-variants following AEX. TheAEX strip was analyzed and the percent oxo-tryptophan species was foundto be about 0.089%. When this strip value was compared to the load (aswell as the flowthrough), it appeared that a significant portion of thisoxo-variant was retained on the AEX column.

The present embodiment can include the addition of one or more steps, inno particular order, such as hydrophobic interaction chromatography,affinity chromatography, multimodal chromatography, viral inactivation(e.g., using low pH), viral filtration, and/or ultra/diafiltration.

One embodiment of the present invention is directed to a method forregenerating a chromatography column comprising a resin. In one aspectof the embodiment, the resin has an immobilized hydrolyzing agent. Inyet another aspect of the embodiment, the resin comprises an immobilizedprotease enzyme. In still another aspect of the embodiment, the resin isa FabRICATOR® resin or a mutant of the resin. In one aspect of theembodiment, the method of regenerating a column comprising a resinimproves reaction efficiency of the resin.

In one aspect of the embodiment, a method of regenerating a columncomprising a resin includes incubating the column resin with aceticacid. In one aspect, the concentration of acetic acid used is from about0.1 M to about 2 M. In one aspect, the concentration of acetic acid isabout 0.5 M. In one aspect, the resin is incubated for at least about 10minutes. In another aspect, the resin is incubated for at least about 30minutes. In yet another aspect of this embodiment, the resin isincubated for at least about 50 minutes. In yet another aspect of thisembodiment, the resin is incubated for at least about 100 minutes. Inyet another aspect of this embodiment, the resin is incubated for atleast about 200 minutes. In yet another aspect of this embodiment, theresin is incubated for at least about 300 minutes.

Optionally, the column resin is further incubated with guanidinehydrochloride (Gu-HCl). In one aspect, Gu-HCl absent acetic acid is usedto regenerate the column resin. The concentration of Gu-HCl employed isfrom about 1 N to about 10 N. In another aspect, the concentration ofGu-HCl is about 6 N. In a further aspect, the column resin can beincubated for at least about 10 minutes with the regenerative agents(acetic acid, Gu-HCl). In yet another aspect, the resin is incubated forat least about 30 minutes. In still another aspect, the resin isincubated for at least about 50 minutes. In yet another aspect, saidresin is incubated for at least about 100 minutes.

In one embodiment, the column comprising a resin is stored in ethanol.In one aspect, the column is stored in ethanol, wherein the ethanolpercentage is from about 5% v/v to about 20% v/v. In a particularaspect, the column is stored using 20% v/v ethanol.

In one embodiment, the process can further comprise formulating the VEGFMiniTrap using a pharmaceutically acceptable excipient. In one aspect,the pharmaceutically acceptable excipient can be selected from thefollowing: water, buffering agents, sugar, salt, surfactant, amino acid,polyol, chelating agent, emulsifier and preservative. Other well-knownexcipients to the skilled artisan are within the purview of thisembodiment.

The formulation of the present invention is suitable for administrationto a human subject. In one aspect of the present embodiment,administration can be effected by intravitreal injection. In one aspect,the formulation can have about 40 to about 200 mg/mL of the protein ofinterest. In a particular aspect, the protein of interest is eitheraflibercept or aflibercept MiniTrap.

The formulation can be used in a method of treating or preventingangiogenic eye disorders which can include: age-related maculardegeneration (e.g., wet or dry), macular edema, macular edema followingretinal vein occlusion, retinal vein occlusion (RVO), central retinalvein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabeticmacular edema (DME), choroidal neovascularization (CNV), irisneovascularization, neovascular glaucoma, post-surgical fibrosis inglaucoma, proliferative vitreoretinopathy (PVR), optic discneovascularization, corneal neovascularization, retinalneovascularization, vitreal neovascularization, pannus, pterygium,vascular retinopathy, diabetic retinopathy in a subject with diabeticmacular edema; or diabetic retinopathies (e.g., non-proliferativediabetic retinopathy (e.g., characterized by a Diabetic RetinopathySeverity Scale (DRSS) level of about 47 or 53) or proliferative diabeticretinopathy; e.g., in a subject that does not suffer from DME).

Variants of IdeS

The present disclosure describes the use of IdeS (FabRICATOR) (SEQ IDNO.: 1) or other polypeptides which are IdeS variants (SEQ ID NO.: 2 to16) to produce a VEGF MiniTrap. IdeS (SEQ ID NO.: 1) includes asparagineresidues at position 87, 130, 182 and/or 274 (shown as “N*” bolded anditalicized in SEQ ID NO.: 1 below). The asparagine at these positionsmay be mutated to an amino acid other than asparagine to form IdeSvariants (and the mutated amino acid(s) are shown as italicized andunderscored amino acid(s)):

SEQ ID NO.: 1MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

*GKDDLLCGAATAGNMLHWWFDQNKDQI KRYLEEHPEKQKINF

*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVF PDHVIDMFI

*GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

*GNLKAIYVTDSDSNAS IGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 2MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

GKDDLLCGAATAGNMLHWWFDQNKDQIK RYLEEHPEKQKINF

*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFP DHVIDMFI

*GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

*GNLKAIYVTDSDSNASI GMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 3MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

*GKDDLLCGAATAGNMLHWWFDQNKDQI KRYLEEHPEKQKINF

GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFP DHVIDMFI

*GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

*GNLKAIYVTDSDSNASI GMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 4MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

*GKDDLLCGAATAGNMLHWWFDQNKDQI KRYLEEHPEKQKINF

*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVF PDHVIDMFI

GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

*GNLKAIYVTDSDSNASI GMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 5MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

*GKDDLLCGAATAGNMLHWWFDQNKDQI KRYLEEHPEKQKINF

*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVF PDHVIDMFI

*GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

GNLKAIYVTDSDSNASI GMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 6MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

GKDDLLCGAATAGNMLHWWFDQNKDQIK RYLEEHPEKQKINF

GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPD HVIDMFI

*GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

*GNLKAIYVTDSDSNASIG MKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 7MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

GKDDLLCGAATAGNMLHWWFDQNKDQIK RYLEEHPEKQKINF

*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFP DHVIDMFI

GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

*GNLKAIYVTDSDSNASIG MKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 8MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

GKDDLLCGAATAGNMLHWWFDQNKDQIK RYLEEHPEKQKINF

*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFP DHVIDMFI

*GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

GNLKAIYVTDSDSNASIG MKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 9MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

*GKDDLLCGAATAGNMLHWWFDQNKDQI KRYLEEHPEKQKINF

GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFP DHVIDMFI

GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

*GNLKAIYVTDSDSNASIG MKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 10MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

*GKDDLLCGAATAGNMLHWWFDQNKDQI KRYLEEHPEKQKINF

GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFP DHVIDMFI

*GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

GNLKAIYVTDSDSNASIG MKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 11MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

*GKDDLLCGAATAGNMLHWWFDQNKDQI KRYLEEHPEKQKINF

*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVF PDHVIDMFI

GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

GNLKAIYVTDSDSNASIG MKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 12MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

GKDDLLCGAATAGNMLHWWFDQNKDQIK RYLEEHPEKQKINF

GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPD HVIDMFI

GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

*GNLKAIYVTDSDSNASIGM KKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 13MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

GKDDLLCGAATAGNMLHWWFDQNKDQIK RYLEEHPEKQKINF

GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPD HVIDMFI

*GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

GNLKAIYVTDSDSNASIGM KKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 14MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

GKDDLLCGAATAGNMLHWWFDQNKDQIK RYLEEHPEKQKINF

*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFP DHVIDMFI

GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

GNLKAIYVTDSDSNASIGM KKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 15MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

*GKDDLLCGAATAGNMLHWWFDQNKDQI KRYLEEHPEKQKINF

GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFP DHVIDMFI

GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

GNLKAIYVTDSDSNASIGM KKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTNSEQ ID NO.: 16MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF

GKDDLLCGAATAGNMLHWWFDQNKDQIK RYLEEHPEKQKINF

GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPD HVIDMFI

GYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS

GNLKAIYVTDSDSNASIGMK KYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

In one embodiment, the polypeptide has an isolated amino acid sequencecomprising at least 70% sequence identity over a full length of anisolated amino acid sequence as set forth in the group consisting of SEQID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6,SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.:11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 andSEQ ID NO.: 16. In one aspect, the isolated amino acid sequence has atleast about 80% sequence identity over a full length of the isolatedamino acid sequence. In another aspect, the isolated amino acid sequencehas at least about 90% sequence identity over a full length of theisolated amino acid sequence. In another aspect, the isolated amino acidsequence has about 100% sequence identity over a full length of theisolated amino acid sequence. In one aspect, the polypeptide can becapable of cleaving a target protein into fragments. In a particularaspect, the target protein is an IgG. In another aspect, the targetprotein is a fusion protein. In yet another aspect, the fragments cancomprise a Fab fragment and/or an Fc fragment.

The present disclosure also includes an isolated nucleic acid moleculeencoding a polypeptide having an isolated amino acid sequence comprisingat least 70% sequence identity over a full length of the isolated aminoacid sequence as set forth in the group consisting of SEQ ID NO.: 2, SEQID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7,SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ IDNO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.:16. In one aspect, the isolated amino acid sequence has at least about80% sequence identity over a full length of the isolated amino acidsequence. In another aspect, the isolated amino acid sequence has atleast about 90% sequence identity over a full length of the isolatedamino acid sequence. In another aspect, the isolated amino acid sequencehas about 100% sequence identity over a full length of the isolatedamino acid sequence. In one aspect, the polypeptide can be capable ofcleaving a target protein into fragments. In a particular aspect, thetarget protein is an IgG. In another particular aspect, the targetprotein is a fusion protein. In yet another particular aspect, thefragments can comprise a Fab fragment and/or an Fc fragment.

The present disclosure also includes a vector which comprises a nucleicacid encoding a polypeptide having an isolated amino acid sequencecomprising at least 70% sequence identity over a full length of theisolated amino acid sequence as set forth in the group consisting of SEQID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6,SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.:11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 andSEQ ID NO.: 16. In one aspect, the nucleic acid molecule is operativelylinked to an expression control sequence capable of directing itsexpression in a host cell. In one aspect, the vector can be a plasmid.In one aspect, the isolated amino acid sequence has at least about 80%sequence identity over a full length of the isolated amino acidsequence. In another aspect, the isolated amino acid sequence has atleast about 90% sequence identity over a full length of the isolatedamino acid sequence. In another aspect, the isolated amino acid sequencehas about 100% sequence identity over a full length of the isolatedamino acid sequence. In one aspect, the polypeptide can be capable ofcleaving a target protein into fragments. In a particular aspect, thetarget protein is an IgG. In another aspect, the target protein is afusion protein. In yet another aspect, the fragments can comprise a Fabfragment and/or an Fc fragment.

In one embodiment, the isolated amino acid can comprise a parental aminoacid sequence defined by SEQ ID NO.: 1 with an asparagine residue atposition 87, 130, 182 and/or 274 mutated to an amino acid other thanasparagine. In one aspect, the mutation can confer an increased chemicalstability at alkaline pH-values compared to the parental amino acidsequence. In another aspect, the mutation can confer an increase inchemical stability by 50% at alkaline pH-values compared to the parentalamino acid sequence. In one aspect, the amino acid can be selected fromaspartic acid, leucine, and arginine. In a particular aspect, theasparagine residue at position 87 is mutated to an aspartic acidresidue. In another aspect, the asparagine residue at position 130 ismutated to an arginine residue. In yet another aspect, the asparagineresidue at position 182 is mutated to a leucine residue. In yet anotheraspect, the asparagine residue at position 274 is mutated to an asparticacid residue. In yet another aspect, the asparagine residues atpositions 87 and 130 are mutated. In yet another aspect, the asparagineresidues at positions 87 and 182 are mutated. In yet another aspect, theasparagine residues at positions 87 and 274 are mutated. In yet anotheraspect, the asparagine residues at positions 130 and 182 are mutated. Inyet another aspect, the asparagine residues at positions 130 and 274 aremutated. In yet another aspect, the asparagine residues at positions 182and 274 are mutated. In yet another aspect, the asparagine residues atpositions 87, 130 and 182 are mutated. In yet another aspect, theasparagine residues at positions 87, 182 and 274 are mutated. In yetanother aspect, the asparagine residues at positions 130, 182 and 274are mutated. In yet another aspect, the asparagine residues at positions87, 130, 182 and 274 are mutated.

In a related embodiment, the disclosure includes an isolated nucleicacid molecule encoding a polypeptide having an isolated amino acidsequence comprising a parental amino acid sequence defined by SEQ IDNO.: 1 with asparagine residues at positions 87, 130, 182 and/or 274mutated to an amino acid other than asparagine—see above. The mutationcan confer an increased chemical stability at alkaline pH-valuescompared to the parental amino acid sequence.

In a further related embodiment, the disclosure includes a vector, whichcomprises a nucleic acid molecule encoding a polypeptide having anisolated amino acid sequence comprising a parental amino acid sequencedefined by SEQ ID NO.: 1 with an asparagine residue at position 87, 130,182 and/or 274 mutated to an amino acid other than asparagine—see above.The mutation can confer an increased chemical stability at alkalinepH-values compared to the parental amino acid sequence. In one aspect,the nucleic acid molecule is operatively linked to an expression controlsequence capable of directing its expression in a host cell. In oneaspect, the vector can be a plasmid.

Affinity-Based Production

The present disclosure also provides methods for reducing host cellproteins as well as other undesirable proteins and nucleic acids duringproduction of an anti-VEGF protein using affinity chromatography.

In one embodiment, a method of producing a recombinant proteincomprises: (a) providing a host cell genetically engineered to express arecombinant protein of interest; (b) culturing the host cell undersuitable conditions in which the cell expresses the recombinant proteinof interest; and (c) harvesting a preparation of the recombinant proteinof interest produced by the cell. In one aspect, the recombinant proteinof interest is an anti-VEGF protein. In a particular aspect, theanti-VEGF protein is selected from the group consisting of aflibercept,MiniTrap, recombinant MiniTrap (an example of which is disclosed in U.S.Pat. No. 7,279,159), a scFv and other anti-VEGF proteins.

In one aspect of the present embodiment, the recombinant protein ofinterest is expressed in a suitable host cell. Non-limiting examples ofsuitable host cells include, but are not limited to, CHO, CHO K1,EESYR®, NICE®, NSO, Sp2/0, embryonic kidney cells and BHK.

In one aspect of the present embodiment, the recombinant protein ofinterest is cultured in a CDM. A suitable CDM includes Dulbecco'sModified Eagle's (DME) medium, Ham's Nutrient Mixture, Excell medium, ISCHO-CD medium, and CDM1B. Other CDMs known to those skilled in the artare also contemplated to be within the scope of the present invention.

The production preparation can comprise at least one contaminantincluding one or more host cell proteins in addition to the recombinantprotein of interest. The at least one contaminant can be derived fromcell-substrate, cell culture or a downstream process.

In one embodiment, the invention is directed to methods for producing ananti-VEGF protein from a biological sample using affinitychromatography. In a particular aspect, methods disclosed herein can beused to separate, at least in part, the anti-VEGF protein from one ormore host cell proteins and nucleic acids (e.g., DNA) formed during theculture production process of an anti-VEGF protein.

In one aspect, the method can comprise subjecting a biological samplecomprising the anti-VEGF protein along with accompanying contaminants toan affinity chromatography under suitable conditions. In a particularaspect, the affinity chromatography can comprise material capable ofselectively or specifically binding to the anti-VEGF protein(“capture”). Non-limiting examples of such chromatographic materialinclude: Protein A, Protein G, chromatographic material comprising, forexample, protein capable of binding to the anti-VEGF protein, andchromatographic material comprising an Fc binding protein. In a specificaspect, the protein capable of binding to or interacting with theanti-VEGF protein can be an antibody, fusion protein or a fragmentthereof. Non-limiting examples of such material capable of selectivelyor specifically binding to the anti-VEGF protein are described inExample 7.

In one aspect of the present embodiment, the method can comprisesubjecting a biological sample comprising an anti-VEGF protein and oneor more host cell proteins/contaminants to affinity chromatography undersuitable conditions, wherein the affinity chromatography stationaryphase comprises a protein capable of selectively or specifically bindingto the anti-VEGF protein. In a particular aspect, the protein can be anantibody, a fusion protein, a scFv or an antibody fragment. In aspecific aspect, the protein can be VEGF₁₆₅, VEGF121, or VEGF forms fromother species, such as rabbit. For example, as exemplified in Table 7-1and Table 7-10, using VEGF165 as the protein capable of selectively orspecifically binding to or interacting with the anti-VEGF protein led toa successful production of MT5 (an anti-VEGF protein), aflibercept andan anti-VEGF scFv fragment. In another specific aspect, the protein canbe one or more of the proteins having an amino acid sequence as shown inSEQ ID NO.: 73-80. Table 7-1 also discloses successful production of MT5using the proteins having amino acid sequences as shown in SEQ ID NO.:73-80 as the protein capable of selectively or specifically binding tothe anti-VEGF protein (MT5).

In one aspect of the present embodiment, the method can comprisesubjecting a biological sample comprising the anti-VEGF protein and oneor more host cell proteins/contaminants to affinity chromatography undersuitable conditions, wherein the affinity chromatography stationaryphase comprises a protein capable of selectively or specifically bindingto or interacting with the anti-VEGF protein, wherein the anti-VEGFprotein can be selected from aflibercept, VEGF MiniTrap, or an anti-VEGFantibody. In a particular aspect, the VEGF MiniTrap can be obtained fromVEGF receptor components; further, it can be formed by recombinantexpression of the VEGF MiniTrap in a host cell. Performing the methodcan reduce the amount of the one or more host cell proteins in thesample. For example, FIG. 35A and FIG. 35B show a significant reductionin total host cell proteins in the sample comprising MT5 (an anti-VEGFprotein) on using five different affinity chromatography columnscomprising (i) VEGF165 (SEQ ID NO.: 72); (ii) mAb1 (a mouse anti-VEGFR1mAb human IgG1 where SEQ ID NO.: 73 is a heavy chain and SEQ ID NO.: 74is a light chain); (iii) mAb2 (a mouse anti-VEGFR1 mAb human IgG1 whereSEQ ID NO.: 75 is a heavy chain and SEQ ID NO.: 76 is a light chain);(iv) mAb3 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 77 is aheavy chain and SEQ ID NO.: 78 is a light chain) and (v) mAb4 (a mouseanti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 79 is a heavy chain and SEQID NO.: 80 is a light chain) as different proteins capable ofselectively or specifically binding to MT5. As seen in FIG. 35A and FIG.35B, the eluates from each of the affinity-based production processesreduced the host cell proteins from above 7000 ppm to about 25 ppm andto about 55 ppm, respectively.

Suitable conditions for employing affinity chromatography can include,but are not limited to, equilibration of an affinity chromatographycolumn using an equilibration buffer. Following equilibration using, forexample, Tris hydrochloride at a pH of about 8.3 to about 8.6, theaffinity chromatography column is loaded with a biological sample.Following loading of the column, the column can be washed one ormultiple times using, for example, the equilibrating buffer such asDulbecco's Phosphate-Buffered Saline (DPBS). Other washes includingwashes employing different buffers can be used before eluting thecolumn. Column elution can be affected by the buffer type and pH andconductivity. Other elution conditions well known to those skilled inthe art can be applied. Following elution using one or more types ofelution buffers, for example, glycine at a pH of about 2.0 to about 3.0,the eluted fractions can be neutralized with the addition of aneutralizing buffer, for example, 1 M Tris at pH 7.5.

In one aspect of the embodiment, the pH of both the wash andequilibration buffer can be from about 7.0 to about 8.6. In one aspectof the embodiment, the wash buffer can be DPBS. In one aspect, theelution buffer can comprise 100 mM glycine buffer with pH of about 2.5.In another aspect, the elution buffer can be a buffer with a pH of about2.0 to about 3.0. In one aspect, the neutralizing buffer can comprise 1M tris with pH of about 7.5.

In one aspect of the present embodiment, the method can further comprisewashing the column with a wash buffer. In one aspect of the presentembodiment, the method can further comprise eluting the column with anelution buffer to obtain elution fractions. In a particular aspect, theamount of host cell proteins in the eluted fractions is significantlyreduced as compared to the amount of host cell proteins in thebiological sample, for example, by about 70%, about 80%, 90%, about 95%,about 98%, or about 99%.

The present embodiment can include the addition of one or more steps, inno particular order, such as hydrophobic interaction chromatography,affinity-based chromatography, multimodal chromatography, viralinactivation (e.g., using low pH), viral filtration, and/orultra/diafiltration.

In one aspect, the glycosylation profile of a composition of ananti-VEGF protein is as follows: about 40% to about 50% totalfucosylated glycans, about 30% to about 55% total sialylated glycans,about 6% to about 15% mannose-5, and about 60% to about 79%galactosylated glycans.

In one aspect of this embodiment, the anti-VEGF protein has Man5glycosylation at about 32.4% of asparagine 123 residues and/or about27.1% of asparagine 196 residues. In a specific embodiment, theanti-VEGF protein can be aflibercept, anti-VEGF antibody or VEGFMiniTrap.

In one embodiment, the method can further comprise formulating a drugsubstance using a pharmaceutically acceptable excipient. In one aspect,the pharmaceutically acceptable excipient can be selected from thefollowing: water, buffering agents, sugar, salt, surfactant, amino acid,polyol, chelating agent, emulsifier and preservative. Other well-knownexcipients to the skilled artisan are within the purview of thisembodiment.

In one aspect of the embodiment, the formulation can be suitable foradministration to a human subject. In one aspect of the presentembodiment, administration can be effected by intravitreal injection. Inone aspect, the formulation can have about 40 to about 200 mg/mL of theprotein of interest. In a particular aspect, the protein of interest canbe aflibercept, anti-VEGF antibody or VEGF MiniTrap.

The formulation can be used in a method of treating or preventingangiogenic eye disorders which can include: age-related maculardegeneration (e.g., wet or dry), macular edema, macular edema followingretinal vein occlusion, retinal vein occlusion (RVO), central retinalvein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabeticmacular edema (DME), choroidal neovascularization (CNV), irisneovascularization, neovascular glaucoma, post-surgical fibrosis inglaucoma, proliferative vitreoretinopathy (PVR), optic discneovascularization, corneal neovascularization, retinalneovascularization, vitreal neovascularization, pannus, pterygium,vascular retinopathy, diabetic retinopathy in a subject with diabeticmacular edema; or diabetic retinopathies (e.g., non-proliferativediabetic retinopathy (e.g., characterized by a Diabetic RetinopathySeverity Scale (DRSS) level of about 47 or 53) or proliferative diabeticretinopathy; e.g., in a subject that does not suffer from DME).

Synthesis of Oxo-Species

One embodiment of the present invention is directed to one or moremethods for synthesizing oxidized protein species using light. In oneaspect of the present embodiment, the protein of interest is ananti-VEGF protein. In a particular aspect, the anti-VEGF protein isaflibercept. In another aspect, the anti-VEGF protein is a VEGF MiniTrapincluding recombinant VEGF MiniTrap. In yet another aspect of thepresent embodiment, the anti-VEGF protein is a single-chain variablefragment (scFv).

In one aspect of the present embodiment, a sample comprises a protein ofinterest, for example, aflibercept fusion protein with minimal or nooxo-variants. The sample is photo-stressed to synthesize oxidizedspecies of aflibercept. In a particular aspect, the sample isphoto-stressed by using cool-white light. In another particular aspect,the sample is photo-stressed by using ultraviolet light.

In a specific aspect of the embodiment, a sample comprising afliberceptor another anti-VEGF protein is exposed to cool-white light for about 30hours to about 300 hours resulting in about 1.5 to about 50-foldincrease in modified oligopeptide. These peptides are enzymaticallydigested and analyzed comprising one or more from the group consistingof:

-   DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID    NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19),    TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21),    SDTGRPFVEMYSEIPEIIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.:    23), SDTGRPFVEM*YSEIPEIIHMTEGR (SEQ ID NO.: 64),    SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.:    66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR (SEQ ID NO.: 28),    RIIW*DSR (SEQ ID NO.: 115), IIW*DSRK (SEQ ID NO.: 114),    TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69),    KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32),    wherein H* is a histidine that is oxidized to 2-oxo-histidine,    wherein C* is a cysteine that is carboxymethylated, wherein M* is an    oxidized methionine, wherein W* is an oxidized tryptophan, wherein    Y* is an oxidized tyrosine, and wherein F* is an oxidized    phenylalanine. The digestion can be performed by proteases alluded    to before, for example, trypsin. The oligopeptides can be analyzed    using mass spectrometry.

In a specific aspect of the embodiment, a sample comprising afliberceptor other anti-VEGF protein is exposed to ultraviolet light for about 4hours to about 40 hours resulting in about 1.5 to about 25-fold increasein modified oligopeptide products (obtained on performing digestion)wherein the sample comprises one or more modified oligopeptides selectedfrom the group consisting of:

-   DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID    NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19),    TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21),    SDTGRPFVEMYSEIPEIIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.:    23), SDTGRPFVEM*YSEIPEIIHMTEGR (SEQ ID NO.: 64),    SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.:    66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR (SEQ ID NO.: 28),    RIIW*DSR (SEQ ID NO.: 115), IIW*DSRK (SEQ ID NO.: 114),    TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69),    KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32),    wherein H* is a histidine that is oxidized to 2-oxo-histidine,    wherein C* is a cysteine that is carboxymethylated, wherein M* is an    oxidized methionine, wherein W* is an oxidized tryptophan, wherein    Y* is an oxidized tyrosine, and wherein F* is an oxidized    phenylalanine. The digestion can be performed by proteases alluded    to before, for example, trypsin. The oligopeptides can be analyzed    using mass spectrometry.

Methods to Minimize Yellow-Brown Color

The present disclosure provides methods for reducing yellow-browncoloration during production of aflibercept, MiniTrap or the likeproduced in a CDM.

In one embodiment, the method comprises culturing a host cell in a CDMunder suitable conditions, wherein the host cell expresses a recombinantprotein of interest, and then harvesting a preparation comprising therecombinant protein of interest. In one aspect, the recombinant proteinof interest is an anti-VEGF protein. In a particular aspect, theanti-VEGF protein is selected from the group consisting of aflibercept,MiniTrap, recombinant MiniTrap (examples of which are disclosed in U.S.Pat. No. 7,279,159, which is incorporated herein by reference in itsentirety), a scFv and other anti-VEGF proteins. In one aspect, themethod can produce a preparation of the recombinant protein of interest,wherein the color of the preparation is characterized using the EuropeanBY method or the CIELAB method (b*). Additionally, the presence ofoxo-variants can be analyzed using, for example, LC-MS.

In one aspect of the present embodiment, mitigation conditions includeincreasing or decreasing cumulative concentrations of one or more mediacomponents, for example, amino acids, metals or antioxidants, including,salts and precursors, corresponding to a reduction in color and proteinvariants of aflibercept and VEGF MiniTrap. Non-limiting examples ofamino acids include alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. In a particular aspect, lowering ofcysteine concentration can be effective in reducing the yellow-browncolor of a preparation. Cysteine concentration can also affectoxo-variants.

In one embodiment, the method comprises culturing a host cell in a CDMunder suitable conditions, wherein the host cell expresses a recombinantprotein of interest, such as aflibercept, and harvesting a preparationof the protein of interest produced by the cell, wherein the suitableconditions are obtained, in part, by lowering the cumulativeconcentration of cysteine in the CDM to less than or equal to about 10mM. Examples of suitable media include, but are not limited to, CDM1B,Excell or the like. As used herein, the term “cumulative amount” refersto the total amount of a particular component added to a bioreactor overthe course of the cell culture to form the CDM, including amounts addedat the beginning of the culture (CDM at day 0) and subsequently addedamounts of the component. Amounts of a component added to a seed-trainculture or inoculum prior to the bioreactor production (i.e., prior tothe CDM at day 0) are also included when calculating the cumulativeamount of the component. A cumulative amount is unaffected by the lossof a component over time during the culture (for example, throughmetabolism or chemical degradation). Thus, two cultures with the samecumulative amounts of a component may nonetheless have differentabsolute levels, for example, if the component is added to the twocultures at different times (e.g., if in one culture all of thecomponent is added at the outset, and in another culture the componentis added over time). A cumulative amount is also unaffected by in situsynthesis of a component over time during the culture (for example, viametabolism or chemical conversion). Thus, two cultures with the samecumulative amounts of a given component may nonetheless have differentabsolute levels, for example, if the component is synthesized in situ inone of the two cultures by way of a bioconversion process. A cumulativeamount may be expressed in units such as, for example, grams or moles ofthe component.

As used herein, the term “cumulative concentration” refers to thecumulative amount of a component divided by the volume of liquid in thebioreactor at the beginning of the production batch, including thecontribution to the starting volume from any inoculum used in theculture. For example, if a bioreactor contains 2 liters of cell culturemedium at the beginning of the production batch, and one gram ofcomponent X is added at days 0, 1, 2, and 3, then the cumulativeconcentration after day 3 is 2 g/L (i.e., 4 grams divided by 2 liters).If, on day 4, an additional one liter of liquid not containing componentX were added to the bioreactor, the cumulative concentration wouldremain 2 g/L. If, on day 5, some quantity of liquid were lost from thebioreactor (for example, through evaporation), the cumulativeconcentration would remain 2 g/L. A cumulative concentration may beexpressed in units such as, for example, grams per liter or moles perliter.

In an aspect of this embodiment, the method comprises culturing a hostcell in a CDM under suitable conditions, wherein the host cell expressesa recombinant protein of interest, harvesting a preparation of theprotein produced by the cell, wherein the suitable conditions areobtained by lowering the ratio of cumulative cysteine concentration fromabout 1:10 to 1:29 to a cumulative total amino acid concentration fromabout 1:50 to about 1:30.

In one embodiment, the method comprises (i) culturing a host cell in aCDM under suitable conditions, wherein the host cell expresses arecombinant protein of interest, such as aflibercept, and (ii)harvesting a preparation of the recombinant protein of interest producedby the cell, wherein the suitable conditions are obtained by loweringthe cumulative concentration of iron in the CDM to less than about 55.0μM. In an aspect of this embodiment, the preparation obtained by thismethod shows lesser yellow-brown color than the preparation obtained bya method wherein the cumulative concentration of iron in the CDM is morethan about 55.0 μM.

In one embodiment, the method comprises culturing a host cell in a CDMunder suitable conditions, wherein the host cell expresses a recombinantprotein of interest, such as aflibercept. The method further comprisesharvesting a preparation of the recombinant protein of interest producedby the cell, wherein the suitable conditions are obtained by loweringthe cumulative concentration of copper in the CDM to less than or equalto about 0.8 μM. In an aspect of this embodiment, the preparationobtained by this method shows lesser yellow-brown color than thepreparation obtained by a method wherein the cumulative concentration ofcopper in the CDM is more than about 0.8 μM.

In one embodiment, the method comprises culturing a host cell in a CDMunder suitable conditions, wherein the host cell expresses a recombinantprotein of interest, such as aflibercept, and harvesting a preparationof the recombinant protein of interest produced by the cell, wherein thesuitable conditions are obtained by lowering the cumulativeconcentration of nickel in the CDM to less than or equal to about 0.40μM. In an aspect of this embodiment, the preparation obtained by thismethod shows lesser yellow-brown color than the preparation obtained bya method wherein the cumulative concentration of nickel in the CDM ismore than about 0.40 μM.

In one embodiment, the method comprises culturing a host cell in a CDMunder suitable conditions, wherein the host cell expresses a recombinantprotein of interest, such as aflibercept. The method further comprisesharvesting a preparation of the recombinant protein of interest producedby the cell, wherein the suitable conditions are obtained by loweringthe cumulative concentration of zinc in the CDM to less than or equal toabout 56 μM. In an aspect of this embodiment, the preparation obtainedby this method shows lesser yellow-brown color than the preparationobtained by a method wherein the cumulative concentration of zinc in theCDM is more than about 56 μM.

In one embodiment, the method comprises culturing a host cell in a CDMunder suitable conditions, wherein the host cell expresses a recombinantprotein of interest, such as aflibercept. The method further comprisesharvesting a preparation of the recombinant protein of interest producedby the cell, wherein the suitable conditions are obtained by presence ofantioxidants in the CDM in a cumulative concentration of about 0.001 mMto about 10 mM for a single antioxidant and no more than about 30 mMcumulative concentration if multiple antioxidants are added in said CDM.In an aspect of this embodiment, the preparation obtained by this methodshows lesser yellow-brown color than the preparation obtained by amethod wherein antioxidants are present in the CDM in a cumulativeconcentration of less than about 0.01 mM or above about 100 mM.Non-limiting examples of the antioxidant can be taurine, hypotaurine,glycine, thioctic acid, glutathione, choline chloride, hydrocortisone,Vitamin C, Vitamin E, chelating agents, catalase,S-carboxymethyl-L-cysteine, and combinations thereof. Non-limitingexamples of chelating agents include aurintricarboxylic acid (ATA),deferoxamine (DFO), EDTA and citrate.

In one embodiment, the method comprises culturing a host cell in a CDMunder suitable conditions, wherein the host cell expresses a recombinantprotein of interest, such as aflibercept. The method further comprisesharvesting a preparation of the recombinant protein of interest producedby the cell, wherein the suitable conditions include a CDM with a:cumulative concentration of iron in said CDM that is less than about 55μM, cumulative concentration of copper in said CDM that is less than orequal to about 0.8 μM, cumulative concentration of nickel in said CDMthat is less than or equal to about 0.40 μM, cumulative concentration ofzinc in said CDM that is less than or equal to about 56 μM, cumulativeconcentration of cysteine in said CDM that is less than 10 mM; and/or anantioxidant in said CDM in a concentration of about 0.001 mM to about 10mM for a single antioxidant, and no more than about 30 mM cumulativeconcentration if multiple antioxidants are added in said CDM.

In one aspect of the present embodiment, the preparation obtained fromusing suitable conditions results in a reduction in protein variants ofaflibercept and VEGF MiniTrap to a desired amount of protein variants ofaflibercept and VEGF MiniTrap (referred to as a “target value” ofprotein variants of aflibercept and VEGF MiniTrap). In a further aspectof this embodiment, the preparation obtained from using suitableconditions results in a reduction in color of the preparations to adesired b* value or BY value (referred to as a “target b* value” “targetBY value” respectively) when the preparation of protein, includingvariants of aflibercept and VEGF MiniTrap are normalized to aconcentration of 5 g/L or 10 g/L. In a further aspect of the presentembodiment, the target b* value (or target BY value) and/or target valueof variants can be obtained in a preparation where the titer increasesor does not significantly decrease.

These and other aspects of the invention will be better appreciated andunderstood when considered in conjunction with the following descriptionand the accompanying drawings. The following description, whileindicating various embodiments and numerous specific details thereof, isgiven by way of illustration and not of limitation. Many substitutions,modifications, additions, or rearrangements may be made within the scopeof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts a VEGF MiniTrap generated using an exemplary embodiment,including VEGFR1 (SEQ ID NO.: 34), VEGFR2 (SEQ ID NO.: 36), Hinge domainfragment (SEQ ID NO.: 60) and the cleaved off Fc fragment fromaflibercept (SEQ ID NO.: 113).

FIG. 2 depicts a proposed mechanism for histidine oxidation to2-oxo-histidine (14 Da).

FIG. 3 depicts a proposed mechanism for histidine oxidation to2-oxo-histidine (16 Da).

FIG. 4 depicts a proposed mechanism for oxidation of tryptophan toN-formylkynurenine and kynurenine.

FIG. 5 depicts an exemplary embodiment for production of aflibercept.

FIG. 6 depicts an exemplary embodiment for production of VEGF MiniTrap.

FIG. 7 depicts an exemplary embodiment for production of aflibercept.

FIG. 8 depicts an exemplary embodiment for production of VEGF MiniTrap.

FIG. 9 depicts a chart of calculated BY standards versus b* valuecalculated according to an exemplary embodiment.

FIG. 10 depicts results of an experiment performed to evaluate thepercentage of 2-oxo-histidines and tryptophan oxidation (whereunderscoring represents oxidation of the residue) in oligopeptides fromprotease-digested AEX load and flowthrough, including fragments ofreduced and alkylated aflibercept (SEQ ID NO.: 55), including SEQ ID NOS114-115, 21, 115, 28, 28, 20, 18, 17, 116-117, and 19, respectively, inorder of appearance.

FIG. 11 depicts the relative abundance of the peptides identified fromthe peptide mapping analysis performed using oligopeptides fromprotease-digested AEX load and flowthrough (where underscoringrepresents oxidation of the residue in the peptide sequence), includingfragments of aflibercept (SEQ ID NO.: 55), including SEQ ID NOS 22, 18,21, 19-20, 118-119, and 28-29, respectively, in order of appearance.

FIG. 12A depicts a full-view of the chromatogram chart of absorbanceversus time (minutes) for MT4 and MT1 at 350 nm.

FIG. 12B depicts an expanded-view of the chromatogram chart ofabsorbance versus time (16-30 minutes) for MT4 and MT1 at 350 nm,including SEQ ID NOS 21, 28, and 28, respectively, in order ofappearance.

FIG. 12C depicts an expanded-view of the chromatogram chart ofabsorbance versus time (30-75 minutes) for MT4 and MT1 at 350 nm,including SEQ ID NOS 17, 20, 18, and 19, respectively, in order ofappearance.

FIG. 13 depicts results of an experiment performed to evaluate thepercentage of 2-oxo-histidines (and tryptophan dioxidation) inoligopeptides from protease digested MT1 which has been processed by AEXchromatography and oligopeptides from protease digested MT1 which hasbeen stripped from AEX chromatography, including SEQ ID NOS 21, 28, 17,20, and 18-19, respectively, in order of appearance.

FIG. 14 depicts results of an experiment performed to compare the acidicspecies present in different production lots of MT1 and the acidic acidfractions obtained on performing a strong cation exchange (CEX)chromatography, including SEQ ID NOS 21, 28, 28, 17, 20, and 18-19,respectively, in order of appearance.

FIG. 15 depicts an exemplary method for the enrichment of the acidicspecies and other variants present in cell culture harvest samples usingstrong cation exchange chromatography.

FIG. 16 depicts the fractions from performing strong cation exchangechromatography according to an exemplary embodiment.

FIG. 17 depicts strong cation exchange chromatograms performed accordingto an exemplary embodiment for the MT1 production (prior to anyproduction procedure, <BY3) subjected to CEX and for enriched variantsof desialylated MiniTrap (dsMT1) using a dual salt-pH gradient.

FIG. 18A depicts a 3D chromatogram for unfractionated parental controlcarried out by strong cation exchange chromatography according to anexemplary embodiment.

FIG. 18B depicts a 3D chromatogram for MT1, fraction 1 representing someof the tailing feature for the experiment carried out by strong cationexchange chromatography according to an exemplary embodiment.

FIG. 18C depicts a 3D chromatogram for MT1, fraction 2 feature carriedout by strong cation exchange chromatography according to an exemplaryembodiment.

FIG. 18D depicts a 3D chromatogram for MT1, fraction 3 feature carriedout by strong cation exchange chromatography according to an exemplaryembodiment.

FIG. 18E depicts a 3D chromatogram for MT1, fraction 4 feature carriedout by strong cation exchange chromatography according to an exemplaryembodiment.

FIG. 18F depicts a 3D chromatogram for MT1, fraction 5 feature carriedout by strong cation exchange chromatography according to an exemplaryembodiment.

FIG. 18G depicts a 3D chromatogram for MT1, fraction 6 feature carriedout by strong cation exchange chromatography according to an exemplaryembodiment.

FIG. 1811 depicts a 3D chromatogram for MT1, fraction 7 feature carriedout by strong cation exchange chromatography according to an exemplaryembodiment.

FIG. 19 depicts imaged capillary isoelectric focusing (icIEF)electropherograms performed according to an exemplary embodiment for theMT1 production.

FIG. 20 depicts results of a study correlating the exposure of MT1 coolwhite light or UVA light with the appearance of oxidized amino acidresidues, including SEQ ID NOS 114, 114-115, 21, 115, 28, 28, 28, 17,83, 20, 18, 29, 29, 19, and 22, respectively, in order of appearance.

FIG. 21 depicts the 3D SEC-PDA (size exclusion chromatography coupled tophotodiode array detection) chromatograms on CWL-stressed MT1 withabsorbance at ˜350 nm (see, e.g., circle highlighting ˜350 nm) accordingto an exemplary embodiment where A shows the chromatogram at T=0, Bshows the chromatogram at 0.5× ICH, C shows the chromatogram at 2.0×ICH, and D shows images of MT1 in vials (normalized to 80 mg/mL)stressed by CWL for different time intervals.

FIG. 22 depicts the 3D SEC-PDA chromatograms on UVA-stressed MT1 withabsorbance at ˜350 nm (see, e.g., circle highlighting ˜350 nm) accordingto an exemplary embodiment where A shows the chromatogram at T=0, Bshows the chromatogram at 0.5× ICH, C shows the chromatogram at 2.0×ICH, and D shows images of MT1 in vials (normalized to 80 mg/mL)stressed by UVA for different time intervals.

FIG. 23A depicts A320/280 absorbance ratios quantitated from SEC-PDAchromatograms for the samples stressed using CWL (top panel) and FIG.23B depicts a chart of A320/280 absorbance ratios for size variants inthe samples stressed using CWL (bottom panel), wherein the samples arestressed according to an exemplary embodiment.

FIG. 24A depicts A320/280 absorbance ratios quantitated from SEC-PDAchromatograms for the samples stressed using UVA (top panel) and FIG.24B depicts a chart of A320/280 absorbance ratios for size variants inthe samples stressed using UVA (bottom panel), wherein the samples arestressed according to an exemplary embodiment.

FIG. 25A depicts a scaled estimate of the effect that incubation ofvarious components with aflibercept have on the generation of color (CIEL*, a*, b* predicted b value); and FIG. 25B depicts actual againstpredicted b value plot.

FIG. 26A depicts the effect of CDMs comprising low cysteine and lowmetals on the titer of aflibercept, FIG. 26B depicts effect of CDMscomprising low cysteine and low metals on the viable cell concentration,FIG. 26C depicts effect of CDMs comprising low cysteine and low metalson the viability, FIG. 26D depicts effect of CDMs comprising lowcysteine and low metals on the ammonia, and FIG. 26E depicts effect ofCDMs comprising low cysteine and low metals on the osmolality.

FIG. 27 is a chart showing prediction profile of the color of theharvest (seen as Day 13 b* values) on increasing/decreasingconcentrations of metals and cysteine according to an exemplaryembodiment.

FIGS. 28A and 28B depicts the effect of incubation of various componentswith aflibercept in spent CDM on the generation of color (CIE L*, a*, b*predicted b value) (A); and by a plot of scaled predicted impacts on bvalue.

FIG. 28C depicts the scaled estimated effects of incubation of variouscomponents with aflibercept in CDM on the generation of color (CIE L*,a*, b* predicted b value) in a shake flask culture.

FIG. 28D depicts the effect of incubation of hypotaurine anddeferoxamine mesylate salt (DFO) with aflibercept in spent CDM on thegeneration of color (CIE L*, a*, b* predicted “b” value).

FIG. 28E depicts the effect of incubation of various componentsindividually with aflibercept from shake flask culture on the generationof color (CIE L*, a*, b* predicted “b” value).

FIG. 29 is a chart showing the effect of addition of uridine, manganese,galactose and dexamethasone in CDMs on the titer of the afliberceptproduced.

FIG. 30 is a chart showing the effect of addition of uridine, manganese,galactose and dexamethasone in CDMs on the viability of cells expressingaflibercept, wherein the aflibercept is produced.

FIG. 31 is a chart showing the effect of addition of uridine, manganese,galactose and dexamethasone in CDMs on the viable cell count of cellsexpressing aflibercept, wherein the aflibercept is produced.

FIG. 32 is a chart showing a standard curve of absorbance versus hostcell protein concentrations (ng/mL) prepared using standard host cellprotein solutions from Cygnus 3G (F550).

FIG. 33 is an image of SDS-PAGE analysis performed using non-reducingSDS-PAGE sample buffer.

FIG. 34 is an image of SDS-PAGE analysis performed using reducingSDS-PAGE sample buffer.

FIG. 35A is a chart of total host cell protein detected in loadingsolution, eluted fractions from affinity chromatography columns 1-3comprising VEGF₁₆₅, mAb1 and mAb2, respectively.

FIG. 35B is a chart of total host cell proteins detected in loadingsolution, eluted fractions from affinity chromatography columns 1, 2, 4and 5 comprising VEGF₁₆₅, mAb1, mAb3 and mAb4, respectively.

FIGS. 36A and 36B depicts the SEC profiles of VEGF MiniTrap A before andB after performing affinity chromatography production, respectively.

FIG. 37 depicts a cartoon representation of the kinetic study of VEGFMiniTrap to VEGF₁₆₅, wherein the VEGF MiniTrap constructs studied werefrom before and after performing affinity chromatography productionaccording to some exemplary embodiments.

FIG. 38 depicts SPR sensorgrams from the kinetic study of VEGF MiniTrapto VEGF₁₆₅, wherein the VEGF MiniTrap constructs studied were frombefore and after performing affinity chromatography production accordingto some exemplary embodiments.

FIG. 39 is a chart of total host cell protein detected in loadingsolution, eluted fractions from affinity chromatography columns usedrepeatedly for columns comprising VEGF₁₆₅, mAb1 and mAb2.

FIG. 40 depicts the structure of VEGF MiniTrap MT1 (SEQ ID NO.: 46)according to an exemplary embodiment.

FIG. 41 depicts the structure of VEGF MiniTrap MT6 (SEQ ID NO.: 51)according to an exemplary embodiment.

FIG. 42 depicts Total Ion Chromatograms (TIC) of relative absorbanceversus time (minutes) for native SEC-MS analysis of MT1, MT5 and MT6 anda zoomed view of the low molecular weight region from the TICs.

FIG. 43 depicts deconvoluted mass spectra of the main peak for MT1 andMT5 to confirm the MiniTrap dimer identity with elucidation for somePTMs, with the N-terminal amino acids indicated (SEQ ID NO.: 120).

FIG. 44 depicts a deconvoluted mass spectrum of the main peak for MT6 toconfirm the single chain MiniTrap identity with elucidation for somePTMs.

FIG. 45A depicts a chart of relative absorbance versus time (minutes)for low molecular weight impurities in MT1.

FIG. 45B depicts mass spectra for the low molecular weight impurities inMT1.

FIG. 46 depicts relative absorbance versus time (minutes) for MT1 whichshows absence of the FabRICATOR enzyme which was used to cleaveaflibercept into MT1.

FIG. 47 depicts relative absorbance versus time (minutes) for lowmolecular weight impurities in MT5.

FIG. 48 depicts relative absorbance versus time (minutes) for lowmolecular weight impurities in MT6.

FIG. 49A depicts a chart of absorbance versus time (minutes) obtained onperforming HILIC-UV/MS for VEGF MiniTrap MT6, wherein the chart showsthe elution of main peak at 21 minutes and 0-glycans at around 21.5minutes.

FIG. 49B depicts a mass spectrum obtained on performing HILIC-UV/MS forVEGF MiniTrap MT6 showing the main peak at 47985.8 Da.

FIG. 49C depicts a mass spectra of O-glycans of VEGF MiniTrap MT6obtained on performing HILIC-UV/MS.

FIG. 50 is an image of VEGF MiniTrap dimer wherein the disulfide bridgein the hinge region (SEQ ID NO.: 83, 123, 83, and 123) of the VEGFMiniTrap can be parallel or crossed.

FIG. 51 depicts relative abundance of distribution of glycans observedat Asn36 among MT1, MT5 and MT6. Figure discloses SEQ ID NO.: 121.

FIG. 52 depicts relative abundance of distribution of glycans observedat Asn68 among MT1, MT5 and MT6. Figure discloses SEQ ID NOS 101 and 30,respectively, in order of appearance.

FIG. 53 depicts relative abundance of distribution of glycans observedat Asn123 among MT1, MT5 and MT6. Figure discloses SEQ ID NO.: 82.

FIG. 54 depicts relative abundance of distribution of glycans observedat Asn196 among MT1, MT5 and MT6. Figure discloses SEQ ID NOS 103 and122, respectively, in order of appearance.

FIG. 55 depicts the released N-linked glycan analysis by hydrophilicinteraction chromatography (HILIC) coupled to fluorescence detection andmass spectrometry analysis (full scale and stacked).

FIG. 56 depicts HILIC-FLR chromatograms for MT1, MT5 and MT6.

FIG. 57 depicts the released N-linked glycan analysis by HILIC coupledto fluorescence detection and mass spectrometry analysis (full scale,stacked and normalized).

FIG. 58A is a table of detailed glycan identification and quantificationfrom VEGF MiniTrap samples MT1, MT5 and MT6.

FIG. 58B is a table of detailed glycan identification and quantificationfrom VEGF MiniTrap samples MT1, MT5 and MT6.

FIG. 58C is a table of detailed glycan identification and quantificationfrom VEGF MiniTrap samples MT1, MT5 and MT6.

FIG. 59 depicts an exemplary production procedure for manufacturingMiniTrap according to an exemplary embodiment.

DETAILED DESCRIPTION

Angiogenesis, the growth of new blood vessels from preexistingvasculature, is a highly orchestrated process that is critical forproper embryonic and postnatal vascular development. Abnormal orpathological angiogenesis is a hallmark of cancer and several retinaldiseases where the upregulation of proangiogenic factors, such asvascular endothelial growth factor (VEGF) leads to increases inendothelial proliferation, changes in vasculature morphology, andincreased vascular permeability. Elevated levels of VEGF have been foundin the vitreous fluid and retinal vasculature of patients with variousocular diseases. Blocking VEGF activity has also become the therapy ofchoice for treating DME, wet AMD, CNV, retinal vein occlusions, andother ocular diseases where abnormal angiogenesis is the underlyingetiology.

As used herein, aflibercept is one such anti-VEGF protein comprising anall-human amino acid sequence comprising the second Ig domain of humanVEGFR1 and the third Ig domain of human VEGFR2 expressed as an inlinefusion with a (Fc) of human IgG1. Aflibercept binds all forms of VEGF-A(VEGF) but in addition binds P1GF and VEGF-B. Several other homodimericVEGF MiniTraps have been generated as enzymatically cleaved productsfrom aflibercept or recombinantly expressed directly from host celllines. One such example of a VEGF MiniTrap is shown in FIG. 1. In thisfigure, a terminal lysine is depicted (K); some culture processes removethis terminal lysine while others do not. FIG. 1 illustrates a processwhereby the terminal lysine remains. In general, aflibercept encompassesboth the presence and absence of the terminal lysine.

As demonstrated herein, the present invention, in part, discloses theproduction of anti-VEGF proteins (Example 1) using a CDM. Analysis ofsolutions comprising aflibercept produced using certain CDMsdemonstrated a certain color property, such as an intense yellow-browncolor. The intensity of the solution's color depended upon the CDM used.Not all CDMs examined produced a sample with a distinct yellow-browncolor after the solutions were normalized to a concentration of 5 g/L.

A color, such as yellow-brown, in an injectable therapeutic drugsolution can be an undesirable feature. It may be an important parameteremployed for determining if a drug product satisfies a predeterminedlevel of purity and quality for a particular therapeutic. A color suchas yellow-brown observed along the manufacturing route of a biologic canbe caused by chemical modifications of that biologic, degradationproducts of formulation excipients, or degradation products formedthrough the reaction of the biologic and formulation excipients.However, such information can be valuable for understanding the cause ofthe color change. It can also assist in designing short-term as well aslong-term storage conditions to prevent modifications facilitating sucha color change.

The inventors observed that use of AEX during the production of ananti-VEGF protein solution minimized yellow-brown coloration.Additionally, the inventors discovered that the yellow-brown colorationcan be decreased by modifying the cell culture used to produce arecombinant protein, such as aflibercept or a modified aflibercept likeMiniTrap.

The present invention encompasses anti-VEGF proteins and theirproduction using CDM. Additionally, the present invention is based onthe identification and optimization of upstream and downstream processtechnologies for protein production.

As demonstrated herein, some of the Examples set forth below describethe production of anti-VEGF proteins (Example 1), production of oxidizedspecies of anti-VEGF proteins (Example 4), methods to reduce oxidizedspecies of anti-VEGF proteins by optimizing culture medium (Example 5)and by optimizing production methods (Example 2).

A number of recent patent applications and granted patents purport todescribe various aflibercept species and methods of producing the same,but none describe or suggest the anti-VEGF compositions and methods forproducing the same described herein. See, e.g., U.S. application Ser.No. 16/566,847 to Coherus Biosciences Inc., U.S. Pat. No. 10,646,546 toSam Chun Dang Pharm. Co., Ltd., U.S. Pat. No. 10,576,128 to Formycon AG,International Application No. PCT/US2020/015659 to Amgen Inc., and U.S.Pat. Nos. 8,956,830; 9,217,168; 9,487,810; 9,663,810; 9,926,583; and10,144,944 to Momenta Pharmaceuticals, Inc.

I. Explanation of Selected Terms

Unless described otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Methods and materialssimilar or equivalent to those described herein known to the skilledartisan can be used in the practice of particular embodiments describedherein. All publications mentioned are hereby incorporated by referencein their entirety.

The term “a” should be understood to mean “at least one” and the terms“about” and “approximately” should be understood to permit standardvariation as would be understood by those of ordinary skill in the artand where ranges are provided, endpoints are included.

As used herein, the term “angiogenic eye disorder” means any disease ofthe eye, which is caused by or associated with the growth orproliferation of blood vessels or by blood vessel leakage.

As used herein, the term “chemically defined medium” or “chemicallydefined media” (both abbreviated “CDM”) refers to a synthetic growthmedium in which the identity and concentration of all the ingredientsare defined. Chemically defined media do not contain bacterial, yeast,animal, or plant extracts, animal serum, or plasma, although individualplant or animal-derived components (e.g., proteins, polypeptides, etc.)may be added. Chemically defined media may contain inorganic salts suchas phosphates, sulfates, and the like needed to support growth. Thecarbon source is defined, and is usually a sugar such as glucose,lactose, galactose, and the like, or other compounds such as glycerol,lactate, acetate, and the like. While certain chemically defined culturemedia also use phosphate salts as a buffer, other buffers may beemployed such as sodium bicarbonate, HEPES, citrate, triethanolamine,and the like. Examples of commercially available chemically definedmedia include, but are not limited to, various Dulbecco's ModifiedEagle's (DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), Ham'sNutrient Mixture (Sigma-Aldrich Co; SAFC Biosciences, Inc.), variousEX-CELLs mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various ISCHO-CD mediums (FUJIFILM Irvine Scientific), combinations thereof, andthe like. Methods of preparing chemically defined culture media areknown in the art, for example, in U.S. Pat. Nos. 6,171,825 and6,936,441, WO 2007/077217, and U.S. Patent Application Publication Nos.2008/0009040 and 2007/0212770, the entire teachings of which are hereinincorporated by reference.

As used herein, the term “cumulative amount” refers to the total amountof a particular component added to a bioreactor over the course of thecell culture to form the CDM, including amounts added at the beginningof the culture (CDM at day 0) and subsequently added amounts of thecomponent. Amounts of a component added to a seed-train culture orinoculum prior to the bioreactor production (i.e., prior to the CDM atday 0) are also included when calculating the cumulative amount of thecomponent. A cumulative amount is unaffected by the loss of a componentover time during the culture (for example, through metabolism orchemical degradation). Thus, two cultures with the same cumulativeamounts of a component may nonetheless have different absolute levels,for example, if the component is added to the two cultures at differenttimes (e.g., if in one culture all of the component is added at theoutset, and in another culture the component is added over time). Acumulative amount is also unaffected by in situ synthesis of a componentover time during the culture (for example, via metabolism or chemicalconversion). Thus, two cultures with the same cumulative amounts of agiven component may nonetheless have different absolute levels, forexample, if the component is synthesized in situ in one of the twocultures by way of a bioconversion process. A cumulative amount may beexpressed in units such as, for example, grams or moles of thecomponent.

As used herein, the term “cumulative concentration” refers to thecumulative amount of a component divided by the volume of liquid in thebioreactor at the beginning of the production batch, including thecontribution to the starting volume from any inoculum used in theculture. For example, if a bioreactor contains 2 liters of cell culturemedium at the beginning of the production batch, and one gram ofcomponent X is added at days 0, 1, 2, and 3, then the cumulativeconcentration after day 3 is 2 g/L (i.e., 4 grams divided by 2 liters).If, on day 4, an additional one liter of liquid not containing componentX were added to the bioreactor, the cumulative concentration wouldremain 2 g/L. If, on day 5, some quantity of liquid were lost from thebioreactor (for example, through evaporation), the cumulativeconcentration would remain 2 g/L. A cumulative concentration may beexpressed in units such as, for example, grams per liter or moles perliter.

As used herein, the term “formulation” refers to a protein of interestthat is formulated together with one or more pharmaceutically acceptablevehicles. In one aspect, the protein of interest is aflibercept and/orMiniTrap. In some exemplary embodiments, the amount of protein ofinterest in the formulation can range from about 0.01 mg/mL to about 600mg/mL. In some specific embodiments, the amount of the protein ofinterest in the formulation can be about 0.01 mg/mL, about 0.02 mg/mL,about 0.03 mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL,about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL,about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL,about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL,about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL,about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 100 mg/mL,about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL,about 150 mg/mL, about 160 mg/mL, about 170 mg/mL, about 180 mg/mL,about 190 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL,about 275 mg/mL, about 300 mg/mL, about 325 mg/mL, about 350 mg/mL,about 375 mg/mL, about 400 mg/mL, about 425 mg/mL, about 450 mg/mL,about 475 mg/mL, about 500 mg/mL, about 525 mg/mL, about 550 mg/mL,about 575 mg/mL, or about 600 mg/mL. In some exemplary embodiments, pHof the composition can be greater than about 5.0. In one exemplaryembodiment, the pH can be greater than about 5.0, greater than about5.5, greater than about 6.0, greater than about 6.5, greater than about7.0, greater than about 7.5, greater than about 8.0, or greater thanabout 8.5.

As used herein, the term “database” refers to a bioinformatics tool,which provides for the possibility of searching the uninterpreted MS-MSspectra against all possible sequences in the database(s). Non-limitingexamples of such tools are Mascot (www.matrixscience.com), Spectrum Mill(www.chem.agilent.com), PLGS (www.waters.com), PEAKS(www.bioinformaticssolutions.com), Proteinpilot(download.appliedbiosystems.com//proteinpilot), Phenyx(www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA(www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem(www.thegpm.org/TANDEM/), Protein Prospector(www.prospector.ucsfedu/prospector/mshome.htm), Byonic(www.proteinmetrics.com/products/byonic) or Sequest(fields.scripps.edu/sequest).

As used herein, the term “ultrafiltration” or “UF” can include amembrane filtration process similar to reverse osmosis, usinghydrostatic pressure to force water through a semi-permeable membrane.Ultrafiltration is described in detail in: LEOS J. ZEMAN & ANDREW L.ZYDNEY, MICROFILTRATION AND ULTRAFILTRATION: PRINCIPLES AND APPLICATIONS(1996), the entire teaching of which is herein incorporated. Filterswith a pore size of smaller than 0.1 μm can be used for ultrafiltration.By employing filters having such small pore size, the volume of thesample can be reduced through permeation of the sample buffer throughthe filter while proteins are retained behind the filter.

As used herein, “diafiltration” or “DF” can include a method of usingultrafilters to remove and exchange salts, sugars, and non-aqueoussolvents, to separate free from bound species, to remove lowmolecular-weight material, and/or to cause the rapid change of ionicand/or pH environments. Microsolutes are removed most efficiently byadding solvent to a solution being ultrafiltered at a rate approximatelyequal to the ultrafiltration rate. This washes microspecies from thesolution at a constant volume. In certain exemplary embodiments of thepresent invention, a diafiltration step can be employed to exchangevarious buffers used in connection with the instant invention, forexample, prior to chromatography or other production steps, as well asto remove impurities from the protein preparation. As used herein, theterm “downstream process technology” refers to one or more techniquesused after the upstream process technologies to produce a protein.Downstream process technology includes, for example, production of aprotein product, using, for example, affinity chromatography, includingProtein A affinity chromatography as well as affinity chromatographythat uses a solid phase having a well-defined molecule like VEGF thatcan interact with its cognate like a VEGF receptor (VEGFR), ion exchangechromatography, such as anion or cation exchange chromatography,hydrophobic interaction chromatography, or displacement chromatography.

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector coding for a protein ofinterest has been introduced. It should be understood that such a termis intended to refer not only to a particular subject cell but to aprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. In an embodiment, host cells include prokaryotic andeukaryotic cells selected from any of the kingdoms of life. In oneaspect, eukaryotic cells include protist, fungal, plant and animalcells. In a further aspect, host cells include eukaryotic cells such asplant and/or animal cells. The cells can be mammalian cells, fish cells,insect cells, amphibian cells or avian cells. In a particular aspect,the host cell is a mammalian cell. A wide variety of mammalian celllines suitable for growth in culture are available from the AmericanType Culture Collection (Manassas, Va.) and other depositories as wellas commercial vendors. Cells that can be used in the processes of theinvention include, but not limited to, MK2.7 cells, PER-C6 cells,Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44(Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; Kolkekar etal., 1997, Biochemistry, 36: 10901-10909; and WO 01/92337 A2),dihydrofolate reductase negative CHO cells (CHO/−DHFR, Urlaub andChasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells(U.S. Pat. No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70);monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCCCRL-1651); HEK 293 cells, and Sp2/0 cells, 5L8 hybridoma cells, Daudicells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells,THP-1 cells, Sp2/0 cells, primary epithelial cells (e.g., keratinocytes,cervical epithelial cells, bronchial epithelial cells, trachealepithelial cells, kidney epithelial cells and retinal epithelial cells)and established cell lines and their strains (e.g., human embryonickidney cells (e.g., 293 cells, or 293 cells subcloned for growth insuspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); babyhamster kidney cells (BHK, ATCC CCL-10); mouse sertoli cells (TM4,Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells(HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lungcells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mousemammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells(BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci.,383:44-68); MCR 5 cells; FS4 cells; PER-C6 retinal cells, MDBK (NBL-1)cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells,Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells,LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells,LLC-MK₂ cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1cells, LLC-PK₁ cells, PK(15) cells, GH₁ cells, GH₃ cells, L2 cells,LLC-RC 256 cells, MH₁C₁ cells, XC cells, MDOK cells, VSW cells, andTH-I, B1 cells, or derivatives thereof), fibroblast cells from anytissue or organ (including but not limited to heart, liver, kidney,colon, intestines, esophagus, stomach, neural tissue (brain, spinalcord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue(lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, andfibroblast and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells,Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26cells, MiCl₁ cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells,African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCCCCL-81); DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells,M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells,C₃H/IOTI/2 cells, HSDM₁C₃ cells, KLN205 cells, McCoy cells, Mouse Lcells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK(Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3cells, Indian muntac cells, SIRC cells, C_(II) cells, and Jensen cells,or derivatives thereof) or any other cell type known to one skilled inthe art.

As used herein, the term “host cell proteins” (HCP) includes proteinderived from a host cell and can be unrelated to the desired protein ofinterest. Host cell proteins can be a process-related impurity which canbe derived from the manufacturing process and can include three majorcategories: cell substrate-derived, cell culture-derived and downstreamderived. Cell substrate-derived impurities include, but are not limitedto, proteins derived from a host organism and nucleic acid (host cellgenomic, vector, or total DNA). Cell culture-derived impurities include,but are not limited to, inducers, antibiotics, serum, and other mediacomponents. Downstream-derived impurities include, but are not limitedto, enzymes, chemical and biochemical processing reagents (e.g.,cyanogen bromide, guanidine, oxidizing and reducing agents), inorganicsalts (e.g., heavy metals, arsenic, nonmetallic ion), solvents,carriers, ligands (e.g., monoclonal antibodies), and other leachables.

In some exemplary embodiments, the host cell protein can have a pI inthe range of about 4.5 to about 9.0. In an exemplary embodiment, the pIcan be about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8,about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1,about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4,about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.

As used herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different agents that can performdigestion of a protein. Non-limiting examples of hydrolyzing agents thatcan carry out enzymatic digestion include protease from Aspergillussaitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N,chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysCendoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinaseArg-C (Arg-C), endoproteinase Glu-C (Glu-C) or outer membrane protein T(OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes(IdeS), thermolysin, papain, pronase, V8 protease or biologically activefragments or homologs thereof or combinations thereof. Non-limitingexamples of hydrolyzing agents that can carry out non-enzymaticdigestion include the use of high temperature, microwave, ultrasound,high pressure, infrared, solvents (non-limiting examples are ethanol andacetonitrile), immobilized enzyme digestion (IMER), magnetic particleimmobilized enzymes, and on-chip immobilized enzymes. For a recentreview discussing the available techniques for protein digestion, seeSwitzar et al., “Protein Digestion: An Overview of the AvailableTechniques and Recent Developments” (Linda Switzar, Martin Giera &Wilfried M. A. Niessen, Protein Digestion: An Overview of the AvailableTechniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH1067-1077 (2013), the entire teachings of which are hereinincorporated). One or a combination of hydrolyzing agents can cleavepeptide bonds in a protein or polypeptide, in a sequence-specificmanner, generating a predictable collection of shorter peptides. Theratio of hydrolyzing agent to protein and the time required fordigestion can be appropriately selected to obtain optimal digestion ofthe protein. When the enzyme to substrate ratio is unsuitably high, thecorrespondingly high digestion rate will not allow sufficient time forthe peptides to be analyzed by mass spectrometer, and sequence coveragewill be compromised. On the other hand, a low E/S ratio would need longdigestion and thus long data acquisition time. The enzyme to substrateratio can range from about 1:0.5 to about 1:200. As used herein, theterm “digestion” refers to hydrolysis of one or more peptide bonds of aprotein. There are several approaches to carrying out digestion of aprotein in a biological sample using an appropriate hydrolyzing agent,for example, enzymatic digestion or non-enzymatic digestion. One of thewidely accepted methods for digestion of proteins in a sample involvesthe use of proteases. Many proteases are available and each of them havetheir own characteristics in terms of specificity, efficiency, andoptimum digestion conditions. Proteases refer to both endopeptidases andexopeptidases, as classified based on the ability of the protease tocleave at non-terminal or terminal amino acids within a peptide.Alternatively, proteases also refer to the six distinctclasses—aspartic, glutamic, and metalloproteases, cysteine, serine, andthreonine proteases, as classified based on the mechanism of catalysis.The terms “protease” and “peptidase” are used interchangeably to referto enzymes which hydrolyze peptide bonds.

The term “in association with” indicates that components, an anti-VEGFcomposition of the present invention, along with another agent such asanti-ANG2, can be formulated into a single composition for simultaneousdelivery, or formulated separately into two or more compositions (e.g.,a kit including each component). Components administered in associationwith each another can be administered to a subject at a different timethan when the other component is administered; for example, eachadministration may be given non-simultaneously (e.g., separately orsequentially) at intervals over a given period of time. Separatecomponents administered in association with each other may also beadministered essentially simultaneously (e.g., at precisely the sametime or separated by a non-clinically significant time period) duringthe same administration session. Moreover, the separate componentsadministered in association with each other may be administered to asubject by the same or by a different route, for example, a compositionof aflibercept along with another agent such as anti-ANG2, wherein thecomposition of aflibercept comprises about 15% or less of its variants.

As used herein, the term “liquid chromatography” refers to a process inwhich a biological/chemical mixture carried by a liquid can be separatedinto components as a result of differential distribution of thecomponents as they flow through (or into) a stationary liquid or solidphase. Non-limiting examples of liquid chromatography include reversephase liquid chromatography, ion-exchange chromatography, size exclusionchromatography, affinity chromatography, mixed-mode chromatography orhydrophobic chromatography.

As used herein, “affinity chromatography” can include separationsincluding any method by which two substances are separated based upontheir affinity to a chromatographic material. It can comprise subjectingthe substances to a column comprising a suitable affinitychromatographic media. Non-limiting examples of such chromatographicmedia include, but are not limited to, Protein A resin, Protein G resin,affinity supports comprising an antigen against which a binding molecule(e.g., antibody) was produced, protein capable of binding to a proteinof interest and affinity supports comprising an Fc binding protein. Inone aspect, an affinity column can be equilibrated with a suitablebuffer prior to sample loading. An example of a suitable buffer can be aTris/NaCl buffer, pH around 7.0 to 8.0. A skilled artisan can develop asuitable buffer without undue burden. Following this equilibration, asample can be loaded onto the column. Following the loading of thecolumn, the column can be washed one or multiple times using, forexample, the equilibrating buffer. Other washes, including washesemploying different buffers, can be used before eluting the column. Theaffinity column can then be eluted using an appropriate elution buffer.An example of a suitable elution buffer can be an acetic acid/NaClbuffer, pH around 2.0 to 3.5. Again, the skilled artisan can develop anappropriate elution buffer without undue burden. The eluate can bemonitored using techniques well known to those skilled in the art,including UV. For example, the absorbance at 280 nm can be employed,especially if the sample of interest comprises aromatic rings (e.g.,proteins having aromatic amino acids like tryptophan).

As used herein, “ion exchange chromatography” can refer to separationsincluding any method by which two substances are separated based ondifferences in their respective ionic charges, either on the molecule ofinterest and/or chromatographic material as a whole or locally onspecific regions of the molecule of interest and/or chromatographicmaterial, and thus can employ either cationic exchange material oranionic exchange material. Ion exchange chromatography separatesmolecules based on differences between the local charges of themolecules of interest and the local charges of the chromatographicmaterial. A packed ion-exchange chromatography column or an ion-exchangemembrane device can be operated in a bind-elute mode, a flowthroughmode, or a hybrid mode. After washing the column or the membrane devicewith an equilibration buffer or another buffer, product recovery can beachieved by increasing the ionic strength (i.e., conductivity) of theelution buffer to compete with the solute for the charged sites of theion exchange matrix. Changing the pH and thereby altering the charge ofthe solute can be another way to achieve elution of the solute. Thechange in conductivity or pH may be gradual (gradient elution) orstepwise (step elution). Anionic or cationic substituents may beattached to matrices in order to form anionic or cationic supports forchromatography. Non-limiting examples of anionic exchange substituentsinclude diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) andquaternary amine (Q) groups. Cationic substituents include carboxymethyl(CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate(S). Cellulose ion exchange medias or support can include DE23™, DE32™,DE52™, CM-23™, CM-32™, and CM-52™ are available from Whatman Ltd.Maidstone, Kent, U.K. SEPHADEX®-based and -locross-linked ion exchangersare also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX® andDEAE-, Q-, CM- and S-SEPHAROSE® and SEPHAROSE® Fast Flow, and Capto™ Sare all available from GE Healthcare. Further, both DEAE and CMderivitized ethylene glycol-methacrylate copolymer such as TOYOPEARL™DEAE-6505 or M and TOYOPEARL™ CM-650S or M are available from Toso HaasCo., Philadelphia, Pa., or Nuvia S and UNOSphere™ S from BioRad,Hercules, Calif., Eshmuno® S from EMD Millipore, Mass.

As used herein, the term “hydrophobic interaction chromatography resin”can include a solid phase, which can be covalently modified with phenyl,octyl, butyl or the like. It can use the properties of hydrophobicity toseparate molecules from one another. In this type of chromatography,hydrophobic groups such as, phenyl, octyl, hexyl or butyl can form thestationary phase of a column. Molecules such as proteins, peptides andthe like pass through a HIC (hydrophobic interactive chromatography)column that possess one or more hydrophobic regions on their surface orhave hydrophobic pockets and are able to interact with hydrophobicgroups comprising a HIC's stationary phase. Examples of HIC resins orsupport include Phenyl sepharose FF, Capto Phenyl (GE Healthcare,Uppsala, Sweden), Phenyl 650-M (Tosoh Bioscience, Tokyo, Japan) andSartobind Phenyl (Sartorius corporation, New York, USA).

As used herein, the term “Mixed Mode Chromatography” or “multimodalchromatography” (both “MMC”) includes a chromatographic method in whichsolutes interact with a stationary phase through more than oneinteraction mode or mechanism. MMC can be used as an alternative orcomplementary tool to traditional reversed-phased (RP), ion exchange(IEX) and normal phase chromatography (NP). Unlike RP, NP and IEXchromatography, in which hydrophobic interaction, hydrophilicinteraction and ionic interaction respectively are the dominantinteraction modes, mixed-mode chromatography can employ a combination oftwo or more of these interaction modes. Mixed mode chromatography mediacan provide unique selectivity that cannot be reproduced by single modechromatography. Mixed mode chromatography can also provide potentialcost savings, longer column lifetimes and operation flexibility comparedto affinity-based methods. In some exemplary embodiments, mixed modechromatography media can be comprised of mixed mode ligands coupled toan organic or inorganic support, sometimes denoted a base matrix,directly or via a spacer. The support may be in the form of particles,such as essentially spherical particles, a monolith, filter, membrane,surface, capillaries, etc. In some exemplary embodiments, the supportcan be prepared from a native polymer such as cross-linked carbohydratematerial, such as agarose, agPV, cellulose, dextran, chitosan, konjac,carrageenan, gellan, alginate, etc. To obtain high adsorptioncapacities, the support can be porous and ligands are then coupled tothe external surfaces as well as to the pore surfaces. Such nativepolymer supports can be prepared according to standard methods, such asinverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2),393-398 (1964), the entire teachings of which are herein incorporated).Alternatively, the support can be prepared from a synthetic polymer suchas cross-linked synthetic polymers, for example, styrene or styrenederivatives, divinylbenzene, acrylamides, acrylate esters, methacrylateesters, vinyl esters, vinyl amides and the like. Such synthetic polymerscan be produced according to standard methods, for example, “Styrenebased polymer supports developed by suspension polymerization” (RArshady: Chimica e L'Industria 70(9), 70-75 (1988), the entire teachingsof which are herein incorporated). Porous native or synthetic polymersupports are also available from commercial sources, such as such as GEHealthcare, Uppsala, Sweden.

As used herein, the term “mass spectrometer” includes a device capableof identifying specific molecular species and measuring their accuratemasses. The term is meant to include any molecular detector into which apolypeptide or peptide may be characterized. A mass spectrometer caninclude three major parts: the ion source, the mass analyzer, and thedetector. The role of the ion source is to create gas phase ions.Analyte atoms, molecules, or clusters can be transferred into gas phaseand ionized either concurrently (as in electrospray ionization) orthrough separate processes. The choice of ion source depends on theapplication. In some exemplary embodiments, the mass spectrometer can bea tandem mass spectrometer. As used herein, the term “tandem massspectrometry” includes a technique where structural information onsample molecules is obtained by using multiple stages of mass selectionand mass separation. A prerequisite is that the sample molecules betransformed into a gas phase and ionized so that fragments are formed ina predictable and controllable fashion after the first mass selectionstep. Multistage MS/MS, or MS^(n), can be performed by first selectingand isolating a precursor ion (MS²), fragmenting it, isolating a primaryfragment ion (MS³), fragmenting it, isolating a secondary fragment(MS⁴), and so on, as long as one can obtain meaningful information, orthe fragment ion signal is detectable. Tandem MS has been successfullyperformed with a wide variety of analyzer combinations. Which analyzersto combine for a certain application can be determined by many differentfactors, such as sensitivity, selectivity, and speed, but also size,cost, and availability. The two major categories of tandem MS methodsare tandem-in-space and tandem-in-time, but there are also hybrids wheretandem-in-time analyzers are coupled in space or with tandem-in-spaceanalyzers. A tandem-in-space mass spectrometer comprises an ion source,a precursor ion activation device, and at least two non-trapping massanalyzers. Specific m/z separation functions can be designed so that inone section of the instrument ions are selected, dissociated in anintermediate region, and the product ions are then transmitted toanother analyzer for m/z separation and data acquisition. Intandem-in-time, mass spectrometer ions produced in the ion source can betrapped, isolated, fragmented, and m/z separated in the same physicaldevice. The peptides identified by the mass spectrometer can be used assurrogate representatives of the intact protein and their posttranslational modifications. They can be used for proteincharacterization by correlating experimental and theoretical MS/MS data,the latter generated from possible peptides in a protein sequencedatabase. The characterization includes, but is not limited, tosequencing amino acids of the protein fragments, determining proteinsequencing, determining protein de novo sequencing, locatingpost-translational modifications, or identifying post translationalmodifications, or comparability analysis, or combinations thereof.

As used herein, “Mini-Trap” or “MiniTrap” or “MiniTrap binding molecule”is capable of binding to a VEGF molecule. Such MiniTraps can include (i)chimeric polypeptides as well as (ii) multimeric (e.g., dimeric)molecules comprising two or more polypeptides which are boundnon-covalently, for example, by one or more disulfide bridges. MiniTrapscan be produced through chemical modification, enzymatic activity, orrecombinantly manufactured.

As used herein, “VEGF MiniTrap” or “VEGF MiniTrap binding molecule” canbe a molecule or complex of molecules that binds to VEGF and has one ormore sets of VEGF receptor Ig-like domains (or variants thereof) (e.g.,VEGFR1 Ig domain 2 and/or VEGFR2 Ig domain 3 and/or 4) and a modified orabsent multimerizing component (MC), for example, wherein the MC is amodified immunoglobulin Fc. The modification may be the result ofproteolytic digestion of a VEGF trap (e.g., aflibercept or conbercept)or direct expression of the resulting polypeptide chains with theshortened MC sequence. (See the molecular structure depicted in FIG. 1.)FIG. 1 is a depiction of a VEGF MiniTrap molecule, which is the productof proteolysis of aflibercept with Streptococcus pyogenes IdeS. Thehomodimeric molecule is depicted having an Ig hinge domain fragmentconnected by two parallel disulfide bonds. The VEGFR1 domain, the VEGFR2domain and the hinge domain fragment (MC) is indicated. The point inaflibercept where IdeS cleavage occurs is indicated with a “//”. Thecleaved off Fc fragment from aflibercept is also indicated. A singlesuch chimeric polypeptide, which is not dimerized, may also be a VEGFMiniTrap if it has VEGF binding activity. The term “VEGF MiniTrap”includes a single polypeptide comprising a first set of one or more VEGFreceptor Ig domains (or variants thereof), lacking an MC, but fused witha linker (e.g., a peptide linker) to one or more further sets of one ormore VEGF receptor Ig domains (or variants thereof). The VEGF bindingdomains in a VEGF MiniTrap of the present invention may be identical ordifferent from another (see WO2005/00895, the entire teachings of whichare herein incorporated).

For example, in an embodiment of the invention, the unmodifiedimmunoglobulin Fc domain comprises the amino acid sequence or aminoacids 1-226 thereof:

(SEQ ID NO.: 33; wherein X₁ is L or P and X₂ is A or T)DKTHTCPX₁CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKX₂TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Inhibition of VEGF includes, for example, antagonism of VEGF binding toVEGF receptor, for example, by competition with VEGF receptor for VEGF(e.g., VEGF₁₁₀, VEGF₁₂₁ and/or VEGF₁₆₅) binding. Such inhibition mayresult in inhibition of VEGF-mediated activation of VEGFR, for example,inhibition of luciferase expression in a cell line (e.g., HEK293)expressing chimeric VEGF Receptor (e.g., a homodimer thereof) havingVEGFR extracellular domains fused to IL18Rα and/or IL18Rβ intracellulardomains on the cell surface and also having an NFkB-luciferase-IRES-eGFPreporter gene, for example, the cell lineHEK293/D9/Flt-IL18Rα/Flt-IL18Rβ as set forth herein.

The VEGF receptor Ig domain components of the VEGF MiniTraps of thepresent invention can include:

-   -   (i) one or more of the immunoglobulin-like (Ig) domain 2 of        VEGFR1 (Flt1) (R1D2),    -   (ii) one or more of the Ig domain 3 of VEGFR2 (Flk1 or KDR)        (Flk1D3) (R2D3),    -   (iii) one or more of the Ig domain 4 of VEGFR2 (Flk1 or KDR)        (Flk1D4) (R2D4) and/or    -   (iv) one or more of the Ig domain 3 of VEGFR3 (Flt4) (Flt1D3 or        R3D3).

Immunoglobulin-like domains of VEGF receptors may be referred to hereinas VEGFR “Ig” domains. VEGFR Ig domains which are referenced herein, forexample, R1D2 (which may be referred to herein as VEGFR1(d2)), R2D3(which may be referred to herein as VEGFR2(d3)), R2D4 (which may bereferred to herein as VEGFR2(d4)) and R3D3 (which may be referred toherein as VEGFR3(d3)) are intended to encompass not only the completewild-type Ig domain, but also variants thereof which substantiallyretain the functional characteristics of the wild-type domain, forexample, retain the ability to form a functioning VEGF binding domainwhen incorporated into a VEGF MiniTrap. It will be readily apparent toone of skill in the art that numerous variants of the above Ig domains,which will retain substantially the same functional characteristics asthe wild-type domain, can be obtained.

The present invention provides a VEGF MiniTrap polypeptide comprisingthe following domain structure:

-   -   ((R1D2)-(R2D3))_(a)-linker-((R1D2)-(R2D3))_(b);    -   ((R1D2)-(R2D3)-(R2D4))_(c)-linker-((R1D2)-(R2D3)-(R2D4))_(d);    -   ((R1D2)-(R2D3))_(e)-(MC)_(g);    -   ((R1D2)-(R2D3)-(R2D4))_(f)-(MC)_(g);        wherein,    -   R1D2 is the VEGF receptor 1 (VEGFR1) Ig domain 2 (D2);    -   R2D3 is the VEGFR2 Ig domain 3;    -   R2D4 is the VEGFR2 Ig domain 4;    -   MC is a multimerizing component (e.g., an IgG hinge domain or        fragment thereof, for example from IgG1);    -   linker is a peptide comprising about 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15 or 16 amino acids, for example, (GGGS)_(g) (SEQ ID        NO.: 104);        and,

-   Independently,

-   a=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

-   b=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

-   c=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

-   d=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

-   e=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

-   f=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; and

-   g=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In an embodiment of the invention, R1D2 comprises the amino acidsequence: SDTGRPFVEMYSEIPEIIHMTEGRELVWCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIID (SEQ ID NO.: 34). In oneaspect, the R1D2 lacks the N-terminal SDT.

In an embodiment of the invention, R1D2 comprises the amino acidsequence:

(SEQ ID NO.: 35) PFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT.

In an embodiment of the invention, R2D3 comprises the amino acidsequence:

(SEQ ID NO.: 36) VVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHE K.

In an embodiment of the invention, R2D4 comprises the amino acidsequence:

(SEQ ID NO.: 37) PFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPG.

In an embodiment of the invention, R2D4 comprises the amino acidsequence:

(SEQ ID NO.: 38) FVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPIKSEKQSHVVSLVVYVP.

In an embodiment of the invention, a multimerizing component (MC) foruse in a VEGF MiniTrap is a peptide, for example, a modified Fcimmunoglobulin (e.g., from an IgG1) which is capable of binding toanother multimerizing component. In one aspect, an MC is a modified Fcimmunoglobulin that includes the immunoglobulin hinge region. Forexample, in an embodiment of the invention, an MC is a peptidecomprising one or more (e.g., 1, 2, 3, 4, 5 or 6) cysteines that areable to form one or more cysteine bridges with cysteines in another MC,for example, DKTHTCPPC (SEQ ID NO.: 39), DKTHTCPPCPPC (SEQ ID NO.: 40),DKTHTCPPCPPCPPC (SEQ ID NO.: 41), DKTHTC(PPC)h, wherein h is 1, 2, 3, 4,or 5 (SEQ ID NO.: 105), DKTHTCPPCPAPELLG (SEQ ID NO.: 60),DKTHTCPLCPAPELLG (SEQ ID NO.: 43), DKTHTC (SEQ ID NO.: 44) orDKTHTCPLCPAP (SEQ ID NO.: 45).

The present invention also provides a VEGF MiniTrap polypeptidecomprising the following domain structure:

-   (i) (R1D2)_(a)-(R2D3)_(b)-(MC)_(c); or-   (ii) (R1D2)_(a)-(R2D3)_(b)-(R2D4)_(c)-(MC)_(d);    which may be homodimerized with a second of said polypeptides, for    example, by binding between the MCs of each polypeptide,-   wherein-   (i) said R1D2 domains coordinate;-   (ii) said R2D3 domains coordinate; and/or-   (iii) said R2D4 domains coordinate,    to form a dimeric VEGF binding domain.

In an embodiment of the invention, the VEGF MiniTrap polypeptidecomprises the amino acid sequence:

(SEQ ID NO.: 46; MC underscored) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN₃₆ITVTLKKFPLDTL IPDGKRIIWDSRKGFIISN ₆₈ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLN ₁₂₃CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK N ₁₉₆STFVRVHEK

; (SEQ ID NO.: 47; MC underscored)GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTEVRVHENLSVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPGDKTHTCP LCPAPELLG;(SEQ ID NO.: 48; MC underscored) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN₃₆ITVTLKKFPLDTL IPDGKRIIWDSRKGFIISN ₆₈ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLN ₁₂₃CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK N₁₉₆STFVRVHEKDKTHTCPPC; (SEQ ID NO.: 49; MC underscored)SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN ₃₆ITVTLKKFPLDTL IPDGKRIIWDSRKGFIISN₆₈ATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVVLSPSHGIELSVGEKLVLN₁₂₃CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK N ₁₉₆STFVRVHEK

; (SEQ ID NO.: 50; MC underscored); SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN₃₆ITVTLKKFPLDTL IPDGKRIIWDSRKGFIISN ₆₈ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLN ₁₂₃CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK N ₁₉₆STFVRVHEK

or (SEQ ID NO.: 106) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTC-(PPC)x (MC underscored; wherein x is 1, 2, 3, 4 or 5).As discussed, such polypeptides may be multimerized (e.g., dimerized(e.g., homodimerized)) wherein binding between the polypeptides ismediated via the multimerizing components.

In an embodiment of the invention, the VEGFR1 Ig-like domain 2 of themonomeric VEGF MiniTraps of the present invention have N-linkedglycosylation at N36 and/or N68; and/or an intrachain disulfide bridgebetween C30 and C79; and/or, the VEGFR2 Ig-like domain 3 of themonomeric VEGF MiniTraps of the present invention, have N-linkedglycosylation at N123 and/or N196; and/or an intrachain disulfide bridgebetween C124 and C185.

In an embodiment of the invention, the VEGF MiniTrap comprises thestructure:

-   -   (R1D2)₁-(R2D3)₁-(G₄S)₃-(R1D2)₁-(R2D3)₁ (“(G₄S)₃” disclosed as        SEQ ID NO.: 107);    -   (R1D2)₁-(R2D3)₁-(G₄S)₆-(R1D2)₁-(R2D3)₁ (“(G₄S)₆” disclosed as        SEQ ID NO.: 108);    -   (R1D2)₁-(R2D3)₁-(G₄S)₉-(R1D2)₁-(R2D3)₁ (“(G₄S)₉” disclosed as        SEQ ID NO.: 109); or    -   (R1D2)₁-(R2D3)₁-(G₄S)₁₂-(R1D2)₁-(R2D3)₁ (“(G₄S)₁₂” disclosed as        SEQ ID NO.: 110).

G₄S is -Gly-Gly-Gly-Gly-Ser (SEQ ID NO.: 111)

In an embodiment of the invention, the VEGF MiniTrap comprises the aminoacid sequence:

(i) (SEQ ID NO.: 51; linker underscored)SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK; (iii)(SEQ ID NO.: 52; linker underscored)SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAAS SGLMTKKNSTFVRVHEK;(iv) (SEQ ID NO.: 53; linker underscored)SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (v)(SEQ ID NO.: 54; linker underscored)SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTK KNSTFVRVHEK; or (vi)(SEQ ID NO.: 112) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK-(GGGGS)x-SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMT KKNSTFVRVHEK(wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15). Asdiscussed herein, these polypeptides may comprise a secondary structurewherein like VEGFR Ig domains associate to form an intra-chain VEGFbinding domain (e.g., FIG. 2). In an embodiment of the invention, two ormore of such polypeptides multimerize (e.g., dimerize (e.g.,homodimerize)) wherein the VEGFR Ig domains of each chain associate withlike Ig domains of another chain to form an inter-chain VEGF bindingdomain.

In a certain embodiment of the invention, a VEGF MiniTrap of the presentinvention lacks any significant modification of the amino acid residuesof a VEGF MiniTrap polypeptide (e.g., directed chemical modificationsuch as PEGylation or iodoacetamidation, for example at the N- and/orC-terminus).

In an embodiment of the invention, the polypeptide comprises a secondarystructure wherein like VEGFR Ig domains in a single chimeric polypeptide(e.g., (R1D2)_(a)-(R2D3)_(b)-linker-(R1D2)_(c)-(R2D3)_(d); or(R1D2)_(a)-(R2D3)_(b)-(R2D4)_(c)-linker-(R1D2)_(d)-(R2D3)_(e)-(R2D4)_(f))or in separate chimeric polypeptides (e.g., homodimers) coordinate toform a VEGF binding domain. For example, wherein

-   (i) said R1D2 domains coordinate;-   (ii) said R2D3 domains coordinate; and/or-   (iii) said R2D4 domains coordinate,    to form a VEGF binding domain. FIG. 2 is a description of a single    chain VEGF MiniTrap depicting such domain coordination. The VEGFR1,    VEGFR2 and linker domains are indicated. The linker shown is (G₄S)₆    (SEQ ID NO.: 108). The present invention includes single chain VEGF    MiniTraps with a (G₄S)₃ (SEQ ID NO.: 107); (G₄S)₉ (SEQ ID NO.: 109)    or (G₄S)₁₂ (SEQ ID NO.: 110) linker.

In addition, the present invention also provides a complex comprising aVEGF MiniTrap as discussed herein complexed with a VEGF polypeptide or afragment thereof or fusion thereof. In an embodiment of the invention,the VEGF (e.g., VEGF₁₆₅) is homodimerized and/or the VEGF MiniTrap ishomodimerized in a 2:2 complex (2 VEGFs:2 MiniTraps) and/or VEGFMiniTrap is homodimerized in a 1:1 complex. Complexes can includehomodimerized VEGF molecules bound to homodimerized VEGF MiniTrappolypeptides. In an embodiment of the invention, the complex is in vitro(e.g., immobilized to a solid substrate) or is in the body of a subject.The present invention also includes a composition of complexes of a VEGFdimer (e.g., VEGF₁₆₅) complexed with a VEGF MiniTrap.

As used herein, the term “protein” or “protein of interest” can includeany amino acid polymer having covalently linked amide bonds. Examples ofproteins of interest include, but are not limited to, aflibercept andMiniTrap. Proteins comprise one or more amino acid polymer chains,generally known in the art as “polypeptides.” “Polypeptide” refers to apolymer composed of amino acid residues, related naturally occurringstructural variants, and synthetic non-naturally occurring analogsthereof linked via peptide bonds. “Synthetic peptide or polypeptide”refers to a non-naturally occurring peptide or polypeptide. Syntheticpeptides or polypeptides can be synthesized, for example, using anautomated polypeptide synthesizer. Various solid phase peptide synthesismethods are known to those of skill in the art. A protein may compriseone or multiple polypeptides to form a single functioning biomolecule.In another exemplary aspect, a protein can include antibody fragments,nanobodies, recombinant antibody chimeras, cytokines, chemokines,peptide hormones, and the like. Proteins of interest can include any ofbio-therapeutic proteins, recombinant proteins used in research ortherapy, trap proteins and other chimeric receptor Fc-fusion proteins,chimeric proteins, antibodies, monoclonal antibodies, polyclonalantibodies, human antibodies, and bispecific antibodies. In a particularaspect, the protein of interest is an anti-VEGF fusion protein (e.g.,aflibercept or MiniTrap). Proteins may be produced using recombinantcell-based production systems, such as the insect bacculovirus system,yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cellsand CHO derivatives like CHO-K1 cells). For a recent review discussingbiotherapeutic proteins and their production, see Ghaderi et al.,“Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation” (Darius Ghaderi et al.,Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY ANDGENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teachings ofwhich are herein incorporated). In some exemplary embodiments, proteinscomprise modifications, adducts, and other covalently linked moieties.These modifications, adducts and moieties include, for example, avidin,streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose,neuraminic acid, N-acetylglucosamine, fucose, mannose, and othermonosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein(MBP), chitin binding protein (CBP), glutathione-S-transferase (GST)myc-epitope, fluorescent labels and other dyes, and the like. Proteinscan be classified on the basis of compositions and solubility and canthus include simple proteins, such as globular proteins and fibrousproteins; conjugated proteins, such as nucleoproteins, glycoproteins,mucoproteins, chromoproteins, phosphoproteins, metalloproteins, andlipoproteins; and derived proteins, such as primary derived proteins andsecondary derived proteins.

In some exemplary embodiments, the protein of interest can be arecombinant protein, an antibody, a bispecific antibody, a multispecificantibody, antibody fragment, monoclonal antibody, fusion protein, scFvand combinations thereof.

As used herein, the term “recombinant protein” refers to a proteinproduced as the result of the transcription and translation of a genecarried on a recombinant expression vector that has been introduced intoa suitable host cell. In certain exemplary embodiments, the recombinantprotein can be a fusion protein. In a particular aspect, the recombinantprotein is an anti-VEGF fusion protein (e.g., aflibercept or MiniTrap).In certain exemplary embodiments, the recombinant protein can be anantibody, for example, a chimeric, humanized, or fully human antibody.In certain exemplary embodiments, the recombinant protein can be anantibody of an isotype selected from group consisting of: IgG, IgM,IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments the antibodymolecule is a full-length antibody (e.g., an IgG1) or alternatively theantibody can be a fragment (e.g., an Fc fragment or a Fab fragment).

The term “antibody,” as used herein includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or VH) and a heavy chain constantregion. The heavy chain constant region comprises three domains, CH1,CH2 and CH3. Each light chain comprises a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region comprises one domain (CL1). The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of theinvention, the FRs of the anti-big-ET-1 antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences or maybe naturally or artificially modified. An amino acid consensus sequencemay be defined based on a side-by-side analysis of two or more CDRs. Theterm “antibody,” as used herein, also includes antigen-binding fragmentsof full antibody molecules. The terms “antigen-binding portion” of anantibody, “antigen-binding fragment” of an antibody, and the like, asused herein, include any naturally occurring, enzymatically obtainable,synthetic, or genetically engineered polypeptide or glycoprotein thatspecifically binds an antigen to form a complex. Antigen-bindingfragments of an antibody may be derived, for example, from full antibodymolecules using any suitable standard techniques such as proteolyticdigestion or recombinant genetic engineering techniques involving themanipulation and expression of DNA encoding antibody variable andoptionally constant domains. Such DNA is known and/or is readilyavailable from, for example, commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include, but are notlimited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFvfragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′ fragment,a Fd fragment, and an isolated complementarity determining region (CDR)region, as well as triabodies, tetrabodies, linear antibodies,single-chain antibody molecules, and multi specific antibodies formedfrom antibody fragments. Fv fragments are the combination of thevariable regions of the immunoglobulin heavy and light chains, and ScFvproteins are recombinant single chain polypeptide molecules in whichimmunoglobulin light and heavy chain variable regions are connected by apeptide linker. In some exemplary embodiments, an antibody fragmentcomprises a sufficient amino acid sequence of the parent antibody ofwhich it is a fragment that it binds to the same antigen as does theparent antibody; in some exemplary embodiments, a fragment binds to theantigen with a comparable affinity to that of the parent antibody and/orcompetes with the parent antibody for binding to the antigen. Anantibody fragment may be produced by any means. For example, an antibodyfragment may be enzymatically or chemically produced by fragmentation ofan intact antibody and/or it may be recombinantly produced from a geneencoding the partial antibody sequence. Alternatively, or additionally,an antibody fragment may be wholly or partially synthetically produced.An antibody fragment may optionally comprise a single chain antibodyfragment. Alternatively, or additionally, an antibody fragment maycomprise multiple chains that are linked together, for example, bydisulfide linkages. An antibody fragment may optionally comprise amulti-molecular complex. A functional antibody fragment typicallycomprises at least about 50 amino acids and more typically comprises atleast about 200 amino acids.

The term “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two different heavy chains with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., antigens) or on the same molecule (e.g., on the sameantigen). If a bispecific antibody is capable of selectively binding twodifferent epitopes (a first epitope and a second epitope), the affinityof the first heavy chain for the first epitope will generally be atleast one to two or three or four orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. The epitopes recognized by the bispecific antibody can be on thesame or a different target (e.g., on the same or a different protein).Bispecific antibodies can be made, for example, by combining heavychains that recognize different epitopes of the same antigen. Forexample, nucleic acid sequences encoding heavy chain variable sequencesthat recognize different epitopes of the same antigen can be fused tonucleic acid sequences encoding different heavy chain constant regionsand such sequences can be expressed in a cell that expresses animmunoglobulin light chain.

A typical bispecific antibody has two heavy chains each having threeheavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain, and aCH3 domain, and an immunoglobulin light chain that either does notconfer antigen-binding specificity but that can associate with eachheavy chain, or that can associate with each heavy chain and that canbind one or more of the epitopes bound by the heavy chainantigen-binding regions, or that can associate with each heavy chain andenable binding of one or both of the heavy chains to one or bothepitopes. BsAbs can be divided into two major classes, those bearing anFc region (IgG-like) and those lacking an Fc region, the latter normallybeing smaller than the IgG and IgG-like bispecific molecules comprisingan Fc. The IgG-like bsAbs can have different formats such as, but notlimited to, triomab, knobs into holes IgG (kih IgG), crossMab, orth-FabIgG, Dual-variable domains Ig (DVD-Ig), two-in-one or dual action Fab(DAF), IgG-single-chain Fv (IgG-scFv), or κλ-bodies. The non-IgG-likedifferent formats include tandem scFvs, diabody format, single-chaindiabody, tandem diabodies (TandAbs), Dual-affinity retargeting molecule(DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock(DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecificantibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies,HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the entire teachingsof which are herein incorporated). The methods of producing bsAbs arenot limited to quadroma technology based on the somatic fusion of twodifferent hybridoma cell lines, chemical conjugation, which involveschemical cross-linkers, and genetic approaches utilizing recombinant DNAtechnology. Examples of bsAbs include those disclosed in the followingpatent applications, which are hereby incorporated by reference: U.S.Ser. No. 12/823,838, filed Jun. 25, 2010; U.S. Ser. No. 13/488,628,filed Jun. 5, 2012; U.S. Ser. No. 14/031,075, filed Sep. 19, 2013; U.S.Ser. No. 14/808,171, filed Jul. 24, 2015; U.S. Ser. No. 15/713,574,filed Sep. 22, 2017; U.S. Ser. No. 15/713,569, field Sep. 22, 2017; U.S.Ser. No. 15/386,453, filed Dec. 21, 2016; U.S. Ser. No. 15/386,443,filed Dec. 21, 2016; U.S. Ser. No. 15/22343 filed Jul. 29, 2016; andU.S. Ser. No. 15/814,095, filed Nov. 15, 2017. Low levels of homodimerimpurities can be present at several steps during the manufacturing ofbispecific antibodies. The detection of such homodimer impurities can bechallenging when performed using intact mass analysis due to lowabundances of the homodimer impurities and the co-elution of theseimpurities with main species when carried out using a regular liquidchromatographic method.

As used herein “multispecific antibody” refers to an antibody withbinding specificities for at least two different antigens. While suchmolecules normally will only bind two antigens (i.e., bispecificantibodies, bsAbs), antibodies with additional specificities such astrispecific antibody and KIH Trispecific can also be addressed by thesystem and method disclosed herein.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodycan be derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, by any means available or known in the art.Monoclonal antibodies useful with the present disclosure can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof.

In some exemplary embodiments, the protein of interest can have a pI inthe range of about 4.5 to about 9.0. In one exemplary specificembodiment, the pI can be about 4.5, about 5.0, about 5.5, about 5.6,about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9,about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2,about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about8.9, or about 9.0. In some exemplary embodiments, the types of proteinof interest in the compositions can be more than one.

In some exemplary embodiments, the protein of interest can be producedfrom mammalian cells. The mammalian cells can be of human origin ornon-human origin can include primary epithelial cells (e.g.,keratinocytes, cervical epithelial cells, bronchial epithelial cells,tracheal epithelial cells, kidney epithelial cells and retinalepithelial cells), established cell lines and their strains (e.g., 293embryonic kidney cells, BHK cells, HeLa cervical epithelial cells andPER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCKcells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells,NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RAcells, WISH cells, BS-C-I cells, LLC-MK2 cells, Clone M-3 cells, 1-10cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PKi cells, PK(15) cells,GHi cells, GH3 cells, L2 cells, LLC-RC 256 cells, MHiCi cells, XC cells,MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells,RK-cells, PK-15 cells or derivatives thereof), fibroblast cells from anytissue or organ (including but not limited to heart, liver, kidney,colon, intestines, esophagus, stomach, neural tissue (brain, spinalcord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue(lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, andfibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells,IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempseycells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells,COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells,F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells,NOR-10 cells, C3H/IOTI/2 cells, HSDMiC3 cells, KLN205 cells, McCoycells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L)cells, L-MTK′ (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, andJensen cells, Sp2/0, NSO, NS1 cells or derivatives thereof).

As used herein, the term “protein alkylating agent” refers to an agentused for alkylating certain free amino acid residues in a protein.Non-limiting examples of protein alkylating agents are iodoacetamide(IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM),methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinationsthereof.

As used herein, “protein denaturing” can refer to a process in which thethree-dimensional shape of a molecule is changed from its native state.Protein denaturation can be carried out using a protein denaturingagent. Non-limiting examples of a protein denaturing agent include heat,high or low pH, reducing agents like DTT (see below) or exposure tochaotropic agents. Several chaotropic agents can be used as proteindenaturing agents. Chaotropic solutes increase the entropy of the systemby interfering with intramolecular interactions mediated by non-covalentforces such as hydrogen bonds, van der Waals forces, and hydrophobiceffects. Non-limiting examples for chaotropic agents include butanol,ethanol, guanidinium chloride, lithium perchlorate, lithium acetate,magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea,N-lauroylsarcosine, urea, and salts thereof.

As used herein, the term “protein reducing agent” refers to the agentused for reduction of disulfide bridges in a protein. Non-limitingexamples of the protein reducing agents used to reduce the protein aredithiothreitol (DTT), ß-mercaptoethanol, Ellman's reagent, hydroxylaminehydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphinehydrochloride (TCEP-HCl), or combinations thereof.

As used herein, the term “variant” of a polypeptide (e.g., of a VEGFR Igdomain) refers to a polypeptide comprising an amino acid sequence thatis at least about 70-99.9% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 99.5, 99.9%) identical or similar to a referenced or nativeamino acid sequence of a protein of interest. A sequence comparison canbe performed by, for example, a BLAST algorithm wherein the parametersof the algorithm are selected to give the largest match between therespective sequences over the entire length of the respective referencesequences (e.g., expect threshold: 10; word size: 3; max matches in aquery range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1;conditional compositional score matrix adjustment). Variants of apolypeptide (e.g., of a VEGFR Ig domain) may also refer to a polypeptidecomprising a referenced amino acid sequence except for one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example,missense mutations (e.g., conservative substitutions), nonsensemutations, deletions, or insertions. The following references relate toBLAST algorithms often used for sequence analysis: BLAST ALGORITHMS:Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, S. F., etal., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) NatureGenet. 3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol.266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids Res.25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton,J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J. M. et al.,(1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS:Dayhoff, M. O., et al., “A model of evolutionary change in proteins.” inAtlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M. O.Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.;Schwartz, R. M., et al., “Matrices for detecting distant relationships.”in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3.” M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington,D.C.; Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J.,et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl.Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol.Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc.Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, N.Y.; the entire teachings of which are hereinincorporated.

Some variants can be covalent modifications that polypeptides undergo,either during (co-translational modification) or after(post-translational modification “PTM”) their ribosomal synthesis. PTMsare generally introduced by specific enzymes or enzyme pathways. Manyoccur at the site of a specific characteristic protein sequence (e.g.,signature sequence) within the protein backbone. Several hundred PTMshave been recorded and these modifications invariably influence someaspect of a protein's structure or function (Walsh, G. “Proteins” (2014)second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853,the entire teachings of which are herein incorporated). In certainexemplary embodiments, a protein composition can comprise more than onetype of protein variant of a protein of interest.

Protein variants in the case of aflibercept (and proteins sharingstructural characteristics of aflibercept, for example, one or moreheavy or light chain regions of aflibercept) can comprise, but are notlimited to, oxidation variants which can result from oxidation of one ormore amino acid residues occurring at, for example, histidine, cysteine,methionine, tryptophan, phenylalanine and/or tyrosine residues;deamidation variants which can result from deamidation at asparagineresidues and/or deoxyglucosonation at arginine residues.

With respect to aflibercept (and proteins sharing structuralcharacteristics of aflibercept, for example, one or more heavy or lightchain regions of aflibercept) oxidation variants can comprise oxidationof histidine residue at His86, His110, His145, His209, His95, His19and/or His203 (or equivalent residue positions on proteins sharingcertain structural characteristics of aflibercept); oxidation oftryptophan residues at Trp58 and/or Trp138 (or equivalent residuepositions on proteins sharing certain structural characteristics ofaflibercept); oxidation of tyrosine residues at Tyr64 (or equivalentpositions on proteins sharing certain structural characteristics ofaflibercept); oxidation of phenylalanine residues at Phe44 and/or Phe166(or equivalent residue positions on proteins sharing certain structuralcharacteristics of aflibercept); and/or oxidation of methionine residuesat Met10, Met20, Met163 and/or Met192 (or equivalent residue positionson proteins sharing certain structural characteristics of aflibercept).

With respect to aflibercept (and proteins sharing structuralcharacteristics of aflibercept, for example, one or more heavy or lightchain regions of aflibercept) deamidation variants can comprisedeamidation of asparagine residue at Asn84 and/or Asn99 (or equivalentresidue positions on proteins sharing certain structural characteristicsof aflibercept).

With respect to aflibercept (and proteins sharing structuralcharacteristics of aflibercept for example, one or more heavy or lightchain regions of aflibercept) deoxyglucosonation variant can comprise3-deoxyglucosonation of arginine residue at Argy (or equivalent residueposition on proteins sharing certain structural characteristics ofaflibercept).

Protein variants can include both acidic species and basic species.Acidic species are typically the variants that elute earlier than themain peak from CEX or later than the main peak from AEX, while basicspecies are the variants that elute later than the main peak from CEX orearlier than the main peak from AEX.

As used herein, the terms “acidic species,” “AS,” “acidic region,” and“AR,” refer to the variants of a protein which are characterized by anoverall acidic charge. For example, in recombinant protein preparationssuch acidic species can be detected by various methods, such as ionexchange, for example, WCX-10 HPLC (a weak cation exchangechromatography), or IEF (isoelectric focusing). Acidic species of anantibody may include variants, structure variants, and/or fragmentationvariants. Exemplary variants can include, but are not limited to,deamidation variants, afucosylation variants, oxidation variants,methylglyoxal (MGO) variants, glycation variants, and citric acidvariants. Exemplary structure variants include, but are not limited to,glycosylation variants and acetonation variants. Exemplary fragmentationvariants include any modified protein species from the target moleculedue to dissociation of peptide chain, enzymatic and/or chemicalmodifications, including, but not limited to, Fc and Fab fragments,fragments missing a Fab, fragments missing a heavy chain variabledomain, C-terminal truncation variants, variants with excision ofN-terminal Asp in the light chain, and variants having N-terminaltruncation of the light chain. Other acidic species variants includevariants comprising unpaired disulfides, host cell proteins, and hostnucleic acids, chromatographic materials, and media components.Commonly, acidic species elute earlier than the main peak during CEX orlater than the main peak during AEX analysis (See FIGS. 16 and 17).

In certain embodiments, a protein composition can comprise more than onetype of acidic species variant. For example, but not by way oflimitation, the total acidic species can be categorized based onchromatographic retention time of the peaks appearing. Another examplein which the total acidic species can be categorized can be based on thetype of variant—variants, structure variants, or fragmentation variant.

The term “acidic species” or “AS” does not refer to process-relatedimpurities. The term “process-related impurity,” as used herein, refersto impurities that are present in a composition comprising a protein,but are not derived from the protein itself. Process-related impuritiesinclude, but are not limited to, host cell proteins (HCPs), host cellnucleic acids, chromatographic materials, and media components.

In one exemplary embodiment, the amount of acidic species in theanti-VEGF composition compared to the protein of interest can be at mostabout 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%,3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%,1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%and ranges within one or more of the preceding. Examples of anti-VEGFcompositions are discussed in Section III below. In one aspect, theanti-VEGF composition can comprise an anti-VEGF protein selected fromthe group consisting of aflibercept, recombinant MiniTrap (examples ofwhich are disclosed in U.S. Pat. No. 7,279,159), a scFv and otheranti-VEGF proteins. In a preferred aspect, the recombinant protein ofinterest is aflibercept.

Among the chemical degradation pathways responsible for acidic or basicspecies, the two most commonly observed covalent modifications occurringin proteins and peptides are deamination and oxidation. Methionine,cysteine, histidine, tryptophan, and tyrosine are some of the aminoacids that are most susceptible to oxidation: Met and Cys because oftheir sulfur atoms and His, Trp, and Tyr because of their aromaticrings.

As used herein, the terms “oxidative species,” “OS,” or “oxidationvariant” refer to the variants of a protein formed by oxidation. Suchoxidative species can also be detected by various methods, such as ionexchange, for example, WCX-10 HPLC (a weak cation exchangechromatography), or IEF (isoelectric focusing). Oxidation variants canresult from oxidation occurring at histidine, cysteine, methionine,tryptophan, phenylalanine and/or tyrosine residues. With respect, inparticular, to aflibercept (and proteins sharing structuralcharacteristics of aflibercept e.g., one or more heavy or light chainregions of aflibercept), oxidation variants can comprise oxidation ofhistidine residue at His86, His110, His145, His209, His95, His19 and/orHis203 (or equivalent residue positions on proteins sharing certainstructural characteristics of aflibercept); oxidation of tryptophanresidues at Trp58 and/or Trp138 (or equivalent residue positions onproteins sharing certain structural characteristics of aflibercept);oxidation of tyrosine residues at Tyr64 (or equivalent positions onproteins sharing certain structural characteristics of aflibercept);oxidation of phenylalanine residues at Phe44 and/or Phe166 (orequivalent residue positions on proteins sharing certain structuralcharacteristics of aflibercept); and/or oxidation of methionine residuesat Met10, Met 20, Met163 and/or Met192 (or equivalent residue positionson proteins sharing certain structural characteristics of aflibercept).

In one exemplary embodiment, the amount of oxidative species in theanti-VEGF composition compared to the protein of interest can be at mostabout 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%,3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and rangeswithin one or more of the preceding. Examples of anti-VEGF compositionsare discussed in Section III below. In one aspect, the anti-VEGFcomposition can comprise an anti-VEGF protein selected from the groupconsisting of aflibercept, recombinant MiniTrap (examples of which aredisclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGFproteins. In a preferred aspect, the recombinant protein of interest isaflibercept or MiniTrap.

Cysteine residues may undergo spontaneous oxidation to form eitherintra- or intermolecular disulfide bonds or monomolecular byproductssuch as sulfenic acid.

Histidine residues are also highly sensitive to oxidation throughreaction with their imidazole rings, which can subsequently generateadditional hydroxyl species (Li, S, C Schoneich, and R T. Borchardt.1995. Chemical Instability of Protein Pharmaceuticals: Mechanisms ofOxidation and Strategies for Stabilization. Biotechnol. Bioeng.48:490-500, the entire teaching of which is herein incorporated).Proposed mechanisms for histidine oxidation are highlighted in FIG. 2and FIG. 3. Detailed mechanistic studies are available in Anal. Chem.2014, 86, 4940-4948 and J. Pharm. Biomed. Anal. 21 (2000) 1093-1097, theentire teaching of which is herein incorporated.

Oxidation of methionine can lead to formation of methionine sulfoxide(Li, S, C Schoneich, and RT. Borchardt. 1995. Chemical Instability ofProtein Pharmaceuticals: Mechanisms of Oxidation and Strategies forStabilization. Biotechnol. Bioeng. 48:490-500). The various possibleoxidation mechanisms of the methionine residues have been discussed inthe literature (Brot, N., Weissbach, H. 1982. The biochemistry ofmethionine sulfoxide residues in proteins. Trends Biochem. Sci. 7:137-139, the entire teaching of which is herein incorporated).

Oxidation of tryptophan can give a complex mixture of products. Theprimary products can be N-formylkynurenine and kynurenine along withmono-oxidation, di-oxidation and/or tri-oxidation products (FIG. 4).Peptides bearing oxidized Trp modifications generally exhibit massincreases of 4, 16, 32 and 48 Da, corresponding to the formation ofkynurenine (KYN), hydroxytryptophan (W_(ox1)), andN-formylkynurenine/dihydroxytryptophan (NFK/W_(ox2), referred to also as“doubly oxidized Trp”), trihydroxytryptophan (W_(ox3), referred to alsoas “triply oxidized Trp”), and their combinations, such ashydroxykynurenine (KYN_(ox1), +20 Da). Oxidation to hydroxytryptophan(W_(ox1)) has been discussed in the literature (Mass spectrometricidentification of oxidative modifications of tryptophan residues inproteins: chemical artifact or post-translational modification? J. Am.Soc. Mass Spectrom. 2010 July; 21(7): 1114-1117, the entire teaching ofwhich is herein incorporated). Tryptophan oxidation, but not methionineand histidine oxidation, has been found to produce a color change inprotein products (Characterization of the Degradation Products of aColor-Changed Monoclonal Antibody: Tryptophan-Derived Chromophores.dx.doi.org/10.1021/ac404218t|Anal. Chem. 2014, 86, 6850-6857). Similarto tryptophan, oxidation of tyrosine primarily yields3,4-dihydroxyphenylalanine (DOPA) and dityrosine (Li, S, C Schoneich,and R T. Borchardt. 1995. Chemical Instability of ProteinPharmaceuticals: Mechanisms of Oxidation and Strategies forStabilization. Biotechnol. Bioeng. 48:490-500).

As used herein, the terms “basic species,” “basic region,” and “BR,”refer to the variants of a protein, for example, an antibody orantigen-binding portion thereof, which are characterized by an overallbasic charge, relative to the primary charge variant species presentwithin the protein. For example, in recombinant protein preparations,such basic species can be detected by various methods, such as ionexchange, for example, WCX-10 HPLC (a weak cation exchangechromatography), or IEF (isoelectric focusing). Exemplary variants caninclude, but are not limited to, lysine variants, isomerization ofaspartic acid, succinimide formation at asparagine, methionineoxidation, amidation, incomplete disulfide bond formation, mutation fromserine to arginine, aglycosylation, fragmentation and aggregation.Commonly, basic species elute later than the main peak during CEX orearlier than the main peak during AEX analysis. (Chromatographicanalysis of the acidic and basic species of recombinant monoclonalantibodies. MAbs. 2012 Sep. 1; 4(5): 578-585. doi: 10.4161/mabs.21328,the entire teaching of which is herein incorporated.)

In certain embodiments, a protein composition can comprise more than onetype of basic species variant. For example, but not by way oflimitation, the total basic species can be divided based onchromatographic retention time of the peaks appearing. Another examplein which the total basic species can be divided can be based on the typeof variant—variants, structure variants, or fragmentation variant.

As discussed for acidic species, the term “basic species” does notinclude process-related impurities and the basic species may be theresult of product preparation (referred to herein as“preparation-derived basic species”), or the result of storage (referredto herein as “storage-derived basic species”).

In one exemplary embodiment, the amount of basic species in theanti-VEGF composition compared to the protein of interest can be at mostabout 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%,3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and rangeswithin one or more of the preceding. Examples of anti-VEGF compositionsare discussed in Section III below. In one aspect, the anti-VEGFcomposition can comprise an anti-VEGF protein selected from the groupconsisting of aflibercept, recombinant MiniTrap (examples of which aredisclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGFproteins. In a preferred aspect, the recombinant protein of interest isaflibercept.

As used herein, “sample matrix” or “biological sample” can be obtainedfrom any step of the bioprocess, such as cell culture fluid (CCF),harvested cell culture fluid (HCCF), any step in the downstreamprocessing, drug substance (DS), or a drug product (DP) comprising thefinal formulated product. In some other specific exemplary embodiments,the biological sample can be selected from any step of the downstreamprocess of clarification, chromatographic production, viralinactivation, or filtration. In some specific exemplary embodiments, thedrug product can be selected from manufactured drug product in theclinic, shipping, storage, or handling.

As used herein, the term “subject” refers to a mammal (e.g., rat, mouse,cat, dog, cow, sheep, horse, goat, rabbit), preferably a human in needof prevention and/or treatment of a cancer or an angiogenic eyedisorder. The subject may have cancer or angiogenic eye disorder or bepredisposed to developing cancer or angiogenic eye disorder.

In terms of protein formulation, the term “stable,” as used hereinrefers to the protein of interest within the formulation being able toretain an acceptable degree of chemical structure or biological functionafter storage under exemplary conditions defined herein. A formulationmay be stable even though the protein of interest contained therein doesnot maintain 100% of its chemical structure or biological function afterstorage for a defined amount of time. Under certain circumstances,maintenance of about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% of a protein's structure or function after storage for adefined amount of time may be regarded as “stable.”

The term “treat” or “treatment” refers to a therapeutic measure thatreverses, stabilizes or eliminates an undesired disease or disorder(e.g., an angiogenic eye disorder or cancer), for example, by causingthe regression, stabilization or elimination of one or more symptoms orindicia of such disease or disorder by any clinically measurable degree,for example, with regard to an angiogenic eye disorder, by causing areduction in or maintenance of diabetic retinopathy severity score(DRSS), by improving or maintaining vision (e.g., in best correctedvisual acuity, for example, as measured by an increase in ETDRSletters), increasing or maintaining visual field and/or reducing ormaintaining central retinal thickness and, with respect to cancer,stopping or reversing the growth, survival and/or metastasis of cancercells in the subject. Typically, the therapeutic measure isadministration of one or more doses of a therapeutically effectiveamount of VEGF MiniTrap to the subject with the disease or disorder.

As used herein, the term “upstream process technology,” in the contextof protein preparation, refers to activities involving the productionand collection of proteins from cells during or following the cellculture of a protein of interest. As used herein, the term “cellculture” refers to methods for generating and maintaining a populationof host cells capable of producing a recombinant protein of interest, aswell as the methods and techniques for optimizing the production andcollection of the protein of interest. For example, once an expressionvector has been incorporated into an appropriate host cell, the hostcell can be maintained under conditions suitable for expression of therelevant nucleotide coding sequences, and the collection and productionof the desired recombinant protein.

When using the cell culture techniques of the instant invention, aprotein of interest can be produced intracellularly, in the periplasmicspace, or directly secreted into the medium. In embodiments where theprotein of interest is produced intracellularly, particulatedebris—either host cells or lysed cells (e.g., resulting fromhomogenization) can be removed by a variety of means, including, but notlimited to, centrifugation or ultrafiltration. Where the protein ofinterest is secreted into the medium, supernatants from such expressionsystems can be first concentrated using a commercially available proteinconcentration filter, for example, using an Amicon™ or MilliporePellicon™ ultrafiltration unit. In one aspect, the protein of interestmay be harvested by centrifugation followed by depth filtration and thenaffinity capture chromatography.

As used herein, a “VEGF antagonist” is any protein or peptide that bindsto or interacts with VEGF. Typically, this binding to or interactingwith inhibits the binding of VEGF to its receptors (VEGFR1 and VEGFR2),and/or inhibits the biological signaling and activity of VEGF. VEGFantagonists include molecules which interfere with the interactionbetween VEGF and a natural VEGF receptor, for example, molecules whichbind to VEGF or a VEGF receptor and prevent or otherwise hinder theinteraction between VEGF and a VEGF receptor. Specific exemplary VEGFantagonists include anti-VEGF antibodies (e.g., ranibizumab[LUCENTIS®]), anti-VEGF receptor antibodies (e.g., anti-VEGFR1antibodies, anti-VEGFR2 antibodies and the like), and VEGFreceptor-based chimeric molecules or VEGF-inhibiting fusion proteins(also referred to herein as “VEGF-Traps” or “VEGF MiniTraps”), such asaflibercept, ziv-aflibercept and a protein having an amino acid havingSEQ ID NO.: 60. Other examples of VEGF-Traps are ALT-L9, M710, FYB203and CHS-2020. Additional examples of VEGF-Traps can be found in U.S.Pat. Nos. 7,070,959; 7,306,799; 7,374,757; 7,374,758; 7,531,173;7,608,261; 5,952,199; 6,100,071; 6,383,486; 6,897,294 & 7,771,721, whichare specifically incorporated herein by reference in their entirety.

VEGF receptor-based chimeric molecules include chimeric polypeptideswhich comprise two or more immunoglobulin (Ig)-like domains of a VEGFreceptor such as VEGFR1 (also referred to as Flt1) and/or VEGFR2 (alsoreferred to as Flk1 or KDR), and may also comprise a multimerizingdomain (e.g., an Fc domain which facilitates the multimerization [e.g.,dimerization] of two or more chimeric polypeptides). An exemplary VEGFreceptor-based chimeric molecule is a molecule referred to asVEGFR1R2-FcAC1(a) (also known as aflibercept; marketed under the productname EYLEA®). In certain exemplary embodiments, aflibercept comprisesthe amino acid sequence set forth as

(SEQ ID NO.: 55) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

As used herein, “viral filtration” can include filtration using suitablefilters including, but not limited to, Planova 2ON™, 50 N or BioEx fromAsahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPVfrom Sartorius, or Ultipor DV20 or DV50™ filter from Pall Corporation.It will be apparent to one of ordinary skill in the art to select asuitable filter to obtain desired filtration performance.

II. Color Determination

As used herein, color observed during the production of a recombinantprotein, specifically, an anti-VEGF protein, can be measured by variousmethods. Non-limiting examples include using the iodine color number,hazen color number, gardner color number, lovibond color number, Sayboltcolor number, Mineral oil color number, European pharmacopoeia colornumber, US pharmacopoeia color number, CIE L*, a*, b* (or CIELAB), Klettcolor number, Hess-Ives color number, the yellowness index, ADMI colornumber, and ASBC and EBC brewery color number. Details on such scalescan be found in Application Report No. 3.9 e by Lange, the entireteaching of which is herein incorporated.

Visual color matching on the basis of the European Pharmacopoeia (PhEur) (European Color Standards, see European Pharmacopoeia. Chapter2.2.2. Degree of coloration of liquids. 8^(th) ed. EP, the entireteaching of which is herein incorporated) can include preparing a colorreference solution as described in Ph. Eur. (EP 2.2.2. Degree ofColoration of Liquids 2)—three parent solutions for red (cobaltous (II)chloride), yellow (ferrous (III) chloride) and blue colors (cuprous (II)sulphate) and 1% hydrochloric acid, five color reference solutions foryellow (Y), greenish-yellow (GY), brownish-yellow (BY), brown (B) andred (R) hues are prepared. With these five reference solutions in turn,a total of thirty-seven color reference solutions are prepared (Y1-Y7,GY1-GY7, BY1-BY7, B1-B9 and R1-R7). Each reference solution is clearlydefined in the CIE-Lab color space, for example, by lightness, hue andchroma. Of the seven yellow-brown standards (BY standards), BY1 is thedarkest standard and BY7 is the least dark. Matching a given sample tothat of a BY color standard is typically done under diffused daylight.The compositions of European yellow-brown color standards are describedin Table 1, below.

TABLE 1 Composition of European Brown-Yellow Standards Volumes in mLReference Standard Hydrochloric acid Solution Solution BY (10 g/1 HCl)BY1 100.0 0.0 BY2 75.0 25.0 BY3 50.0 50.0 BY4 25.0 75.0 BY5 12.5 87.5BY6 5.0 95.0 BY7 2.5 97.5 Brownish-Yellow Standard Solution (BY): 10.8g/L FeCl_(3•)6H₂0, 6.0 g/L CoCl_(2•)6H₂O) and 2.5 g/L CuSO_(4•)5H₂O

The test for color of liquids is carried out by comparing a testsolution with a standard color solution. The composition of the standardcolor solution is selected depending on the hue and intensity of thecolor of the test solution. Typically, comparison is carried out inflat-bottomed tubes of colorless, transparent, neutral glass that arematched as closely as possible in internal diameter and in all otherrespects (e.g., tubes of about 12, 15, 16 or 25 mm diameter). Forexample, a comparison can be between 2 or 10 mL of the test solution andstandard color solution. The depth of liquids, for example, can be about15, 25, 40 or 50 mm. The color assigned to the test solution should notbe more intense than that of the standard color. Color comparisons aretypically carried out in diffused light (e.g., daylight) against a whitebackground. Colors can be compared down the vertical axis or horizontalaxis of the tubes.

In contrast to the EP color measurement, the USP Monograph 1061Color-Instrumental Measurement references the use of CIE L*, a*, b* (orCIELAB) color measurement to quantify colors precisely and objectively.A total of twenty color reference solutions (identified sequentially bythe letters A to T) are defined in U.S. Pharmacopoeia. The color of themeasured sample is automatically correlated to the color referencesolutions. This means that the color reference solution that is closestto the sample (i.e., the reference solution with the smallest colordifference ΔE* to the color of the sample) is displayed. The ΔL*, Δa*and Δb* values give the quantitative differences between the L*, a* andb* values of the sample and those of the displayed USP solutions. In theCIE L*a*b* coordinate system, L* represents the degree of lightness of acolor on a scale of 0-100, with 0 being the darkest and 100 thelightest, a* represents the redness or greenness of a color (positivevalues of a* represent red, whereas negative values of a* representgreen), and b* represents the yellowness or blueness of a sample, withpositive values of b* representing yellow and negative values of b*representing blue. Color difference from a standard, or from an initialsample in an evaluation, can be represented by a change in theindividual color components ΔL*, Δa*, and Δb*. The composite change, ordifference in color, can be calculated as a simple Euclidian distance inspace using the formula: dE*=√{square root over ((ΔL*)²+(Δa*)²+(Δb*)²)}.CIE L*, a*, b* color coordinates can be generated, for example, usingthe Hunter Labs UltrascanPro (Hunter Associates Laboratory, Reston, Va.)or on the BYK Gardner LCS IV (BYK-Gardner, Columbia, Md.). For theHunter Labs UltraScan Pro, the Didymium Filter Test can be executed forwavelength calibration. The instrument can be standardized in TTRAN withthe 0.780-inch port insert and DIW before use; thus, establishing thetop (L=100) and bottom (L=0) of the photometric scale using a light trapand black card. See Pack et al., Modernization of Physical Appearanceand Solution Color Tests Using Quantitative Tristimulus Colorimetry:Advantages, Harmonization, and Validation Strategies, J. PharmaceuticalSci. 104: 3299-3313 (2015), the entire teaching of which is hereinincorporated. The color of the BY standards can also be expressed underthe CIE L*, a*, b* color space (“CIELAB” or “CIELab” color space). SeeTable 2.

TABLE 2 Characterization of European Brown-Yellow Color Standards in theCIE L*, a*, b* Color Space Std. L*^(∧) a*^(∧) b*^(∧) L*^(~) a*^(~)b*^(~) BY1 93.95 −2.76 28.55 92.84 −3.16 31.15 BY2 94.76 −2.96 22.6994.25 −3.77 26.28 BY3 96.47 −2.84 16.41 95.92 −3.44 18.52 BY4 97.17−1.94  9.07 97.67 −2.63 10.70 BY5 98.91 −1.19  4.73 98.75 −1.61  5.77BY6 99.47 −0.59  2.09 99.47 −0.71  2.38 BY7 99.37 −0.31  1.13 99.71−0.37  1.17 ^(∧)Reported by Pack et at. ^(~)Measured experimentallyherein-the L* and b* values, for each BY color standard

To enable a high throughput screening for the color assay, thespectrophometric assay method (CIELAB) is a more suitable andquantitative measure than BY color standards. The surrogate assay wasfurther optimized as described in the Example section.

For any of the samples evaluated for color, the protein concentration ofthe test samples must be standardized for protein concentration in thesamples, for example, 5 g/L, 10 g/L and the like for comparison.

III. Anti-VEGF Compositions

There are at least five members of the VEGF family of proteins thatregulate the VEGF signaling pathway: VEGF-A, VEGF-B, VEGF-C, VEGF-D, andplacental growth factor (PlGF). Anti-VEGF compositions can comprise aVEGF antagonist, which specifically interacts with one or more membersof the VEGF family of proteins and inhibits one or more of itsbiological activities, for example, its mitogenic, angiogenic and/orvascular permeability activity.

In one embodiment, a method of producing an anti-VEGF protein comprises:(a) providing a host cell genetically engineered to express theanti-VEGF protein; (b) culturing the host cell in a CDM under conditionssuitable in which the cell expresses the anti-VEGF protein; and (c)harvesting a preparation of the anti-VEGF protein produced by the cell.In one aspect, the anti-VEGF protein is selected from the groupconsisting of aflibercept, recombinant MiniTrap (examples of which aredisclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGFproteins. In a preferred aspect, the recombinant protein of interest isaflibercept.

The inventors discovered that manufacturing anti-VEGF proteins (e.g.,aflibercept) in certain CDMs produced a biological sample exhibiting adistinctive color. The distinct color properties were observed indifferent manufacturing steps and even in the final formulationcomprising the anti-VEGF protein. As observed in Example 9, for theproduction of VEGF MiniTrap, culturing cells in a CDM produced anti-VEGFproteins (e.g., aflibercept) with an intense yellow-brown color. Theaffinity capture step following harvesting also produced an eluateexhibiting a certain color—a yellow-brown color. Further productionsteps using AEX also exhibited a yellow-brown color, however withreduced intensity.

As described in more detail below, color may be assessed using (i) theEuropean Color Standard BY in which a qualitative visual inspection ismade or (ii) a colorimetric assay, CIELAB, which is more quantitativethan the BY system. However, in either case, color assessment betweenmultiple samples was normalized against protein concentration in orderto assure a meaningful assessment/comparison. For example, referring toExample 9, in particular Table 9-2, the Protein A eluate has a b* valueof around 2.52 which corresponds to approximately a BY value of BY5(when measured at a concentration of 5 g/L protein in the Protein Aeluate). If the color of the Protein A eluate is to be compared toanother sample, then the comparison should be made using the sameprotein concentration. Thus, comparing the Protein A eluate to the AEXpool which has a b* value of around 0.74 (when measured at aconcentration of 5 g/L protein in the protein A eluate), the method ofproduction shows a substantial reduction in the yellow-brown color ofthe sample from the Protein A eluate to the AEX pool following AEXchromatography.

Compositions of the present invention can be characterized by ayellow-brown color as discussed herein, for example, no darker/moreintense than the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4,BY4-BY5 or BY5-BY6 and/or having a b* value 17-23, 10-17, 5-10, 3-5, or1-3, wherein the composition comprises about 5 g/L of the anti-VEGFprotein or about 10 g/L of the anti-VEGF protein and wherein thecomposition is obtained as a sample from a clarified harvest or aProtein A eluate of the clarified harvest.

In one embodiment, the compositions of the invention produced using CDMproduces a biological sample having a distinct yellow-brown color,wherein the sample may be characterized by a recognized standard colorcharacterization:

-   (i) no more yellow-brown than European Color Standard BY2;-   (ii) no more yellow-brown than European Color Standard BY3;-   (iii) no more yellow-brown than European Color Standard BY4;-   (iv) no more yellow-brown than European Color Standard BY5;-   (v) between European Color Standard BY2 and BY3;-   (vi) between European Color Standard BY3 and BY4;-   (vii) between European Color Standard BY4 and BY5, wherein the    composition comprises about 5 g/L or about 10 g/L of the anti-VEGF    protein and wherein the composition is obtained as a sample from a    Protein A eluate of a clarified harvest.

In another embodiment, the compositions of the invention produced usinga CDM produces a biological sample having a distinct yellow-brown color,wherein the composition is characterized by a recognized standard colorcharacterization in the CIELAB scale:

-   (i) no more yellow-brown than a b* value of about 22-23;-   (ii) no more yellow-brown than a b* value of about 16-17;-   (iii) no more yellow-brown than a b* value of 9-10;-   (iv) no more yellow-brown than a b* value of 4-5;-   (v) no more yellow-brown than a b* value of 2-3;-   (vi) between b* value of 17-23;-   (vii) between b* value of 10-17;-   (viii) between b* value of 5-10;-   (ix) between b* value of 3-5; or-   (x) between b* value of 1-3, wherein the composition comprises about    5 g/L or about 10 g/L of the anti-VEGF protein and wherein the    composition is obtained as a sample from a Protein A eluate of a    clarified harvest.

In one embodiment, the compositions of the invention produced using CDMcan comprise other species or variants of the anti-VEGF protein. Thesevariants include anti-VEGF protein isoforms that comprise one or moreoxidized amino acid residues collectively referred to as oxo-variants.The enzymatic digestion of such compositions comprising the anti-VEGFprotein and its oxo-variants can comprise one or more of:

-   EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about    0.004-0.013% 2-oxo-histidines,-   QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which comprises about    0.006-0.028% 2-oxo-histidines,-   TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about    0.049-0.085% 2-oxo-histidines,-   DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about    0.057-0.092% 2-oxo-histidines,-   TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.008-0.022%    2-oxo-histidines, and/or-   IIWDSR (SEQ ID NO.: 56) which comprises about 0.185-0.298%    dioxidized tryptophan; or-   EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.008%    2-oxo-histidines,-   QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which comprises about    0.02% 2-oxo-histidines,-   TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about 0.06%    2-oxo-histidines,-   DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.07%    2-oxo-histidines,-   TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.01%    2-oxo-histidines, and/or-   IIWDSR (SEQ ID NO.: 56) which comprises about 0.23%    di-oxo-tryptophans, wherein H* is a histidine that may be oxidized    to 2-oxo-histidine and wherein C* is a cysteine which may be    carboxymethylated. In a particular embodiment, the anti-VEGF protein    is aflibercept. In another embodiment, the anti-VEGF protein is a    VEGF MiniTrap.

In one exemplary embodiment of the invention, the compositions of theinvention can comprise an anti-VEGF protein, wherein no more than about1%, no more than about 0.1%, or about 0.1-1%, 0.2-1%, 0.3-1%, 0.4-1%,0.5-1%, 0.6-1%, 0.7-1%, 0.8-1% or 0.9-1% of histidine residues of theanti-VEGF protein are 2-oxo-histidine. In such compositions, there canbe a heterogeneous population of the anti-VEGF protein variants eachhaving a varying amount of 2-oxo-histidine residues and un-oxidizedhistidine residues. Thus, the percentage of 2-oxo-histidine anti-VEGFprotein in a composition refers to the site-specific 2-oxo-histidinesamong the anti-VEGF molecules divided by total site-specific histidinesin the molecules of the anti-VEGF protein (oxidized plus un-oxidized)times 100. One method to quantitate the level of 2-oxo-histidines in acomposition is to digest the polypeptide with a protease (e.g., Lys-Cand/or trypsin) and analyze the quantity of 2-oxo-histidines in theresulting peptides by, for example, mass spectrometry (MS).

Before digestion of the anti-VEGF protein, cysteine sulfhydryl groupsare blocked by reaction with iodoacetamide (IAM) resulting in a residuerepresented by the following chemical structure:

Such modification protects free thiols from reforming disulfide bridgesand prevents disulfide bond scrambling. The present invention includescompositions (e.g., aqueous compositions) comprising anti-VEGF proteinand its variants which, when modified with IAM and digested withprotease (e.g., Lys-C and trypsin) and analyzed by mass spectrometrycomprise the following peptides:

-   EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about    0.004-0.013% 2-oxo-histidines,-   QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which comprises about    0.006-0.028% 2-oxo-histidines,-   TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about    0.049-0.085% 2-oxo-histidines,-   DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about    0.057-0.092% 2-oxo-histidines,-   TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.008-0.022%    2-oxo-histidines, and/or-   IIWDSR (SEQ ID NO.: 56) which comprises about 0.185-0.298%    dioxidized tryptophan; or-   EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.008%    2-oxo-histidines,-   QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which comprises about    0.02% 2-oxo-histidines,-   TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about 0.06%    2-oxo-histidines,-   DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.07%    2-oxo-histidines,-   TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.01%    2-oxo-histidines, and/or-   IIWDSR (SEQ ID NO.: 56) which comprises about 0.23%    di-oxo-tryptophans,    wherein H* is 2-oxo-histidine and wherein C* is carboxymethylated    cysteine. In one embodiment of the invention, the peptides are    deglycosylated with PNGase F.

The present invention includes compositions comprising anti-VEGFprotein, wherein about 0.1%-10% of all histidines of the anti-VEGFprotein are modified to 2-oxo-histidine. Further, the color of thecomposition is no darker/more intense than, for example, the EuropeanBrown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6, or,alternatively, having a b* value, as characterized using CIE L*, a*, b*,of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the compositioncomprises about 5 g/L or about 10 g/L of the anti-VEGF protein. Thecomposition is obtained either as a sample from a clarified harvest or aProtein A eluate of the clarified harvest. Such compositions can beobtained from the clarified harvest when the harvest material issubjected to a capture chromatography procedure. In one aspect, thecapture step is an affinity chromatography procedure using, for example,a Protein A affinity column. When an affinity sample is analyzed usingliquid chromatography-mass spectrometry (LC-MS), one or more variantsmay be detected.

The present invention includes compositions comprising anti-VEGFprotein, wherein about 0.1%-10% of all tryptophans of the anti-VEGFprotein are modified to kynurenine. Further, the color of thecomposition is no darker/more intense than the European Brown-YellowColor Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b*value, as characterized by CIE L*, a*, b*, of about 17-23, 10-17, 5-10,3-5, or 1-3, wherein the composition comprises about 5 g/L of theanti-VEGF protein or about 10 g/L of the anti-VEGF protein. Thecomposition is obtained as a sample from a clarified harvest or aProtein A eluate of the clarified harvest. Such compositions can beobtained from the clarified harvest when subjected to a capturechromatography procedure. The capture step is an affinity chromatographyprocedure using, for example, a Protein A affinity column. When anaffinity sample is analyzed using liquid chromatography-massspectrometry (LC-MS), one or more of these variants may be detected.

The present invention includes compositions comprising anti-VEGFprotein, wherein about 0.1%-10% of all tryptophans of the anti-VEGFprotein are modified to mono-hydroxyl tryptophan. Further, the color ofthe composition is no darker/more intense than the European Brown-YellowColor Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b*value characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5,or 1-3, wherein the composition comprises about 5 g/L of the anti-VEGFprotein or about 10 g/L of the anti-VEGF protein. The composition isobtained as a sample from a clarified harvest or a Protein A eluate ofthe clarified harvest. Such compositions can be obtained from theclarified harvest when subjected to a capture chromatography procedure.The capture step is an affinity chromatography procedure using, forexample, a Protein A affinity column. When a sample extracted from theaffinity step is analyzed using liquid chromatography-mass spectrometry(LC-MS), one or more of these variants may be detected.

The present invention includes compositions comprising anti-VEGFprotein, wherein about 0.1%-10% of all tryptophans of the anti-VEGFprotein are modified to di-hydroxyl tryptophan. Further, the color is nodarker/more intense than the European Brown-Yellow Color StandardBY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* valuecharacterized using CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or1-3, wherein the composition comprises about 5 g/L of the anti-VEGFprotein or about 10 g/L of the anti-VEGF protein. The composition isobtained as a sample from a clarified harvest or a Protein A eluate ofthe clarified harvest. Such compositions can be obtained from theclarified harvest made using CDM comprising the anti-VEGF protein aswell as its oxo-variants subjected to a capture chromatographyprocedure. The capture step is an affinity chromatography procedureusing, for example, a Protein A affinity column. When a sample extractedfrom the affinity step is analyzed using liquid chromatography-massspectrometry (LC-MS), one or more of these variants may be detected.

The present invention includes compositions comprising anti-VEGFprotein, wherein about 0.1%-10% of all tryptophans of the anti-VEGFprotein are modified to tri-hydroxyl tryptophan. Further, the color ofthe composition is no darker/more intense than the European Brown-YellowColor Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b*value characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5,or 1-3, wherein the composition comprises about 5 g/L of the anti-VEGFprotein or about 10 g/L of the anti-VEGF protein. The composition isobtained as a sample from a clarified harvest or a Protein A eluate ofthe clarified harvest. Such compositions can be obtained using capturechromatography. The capture step is an affinity chromatography procedureusing, for example, a Protein A affinity column. When a sample extractedfrom the affinity is analyzed using liquid chromatography-massspectrometry (LC-MS), one or more of these variants may be detected.

In one embodiment, the compositions of the invention can comprise ananti-VEGF protein, wherein the anti-VEGF protein can comprisemodifications of one or more residues as follows: one or moreasparagines are deamidated; one or more aspartic acids are converted toiso-aspartate and/or asparagine; one or more methionines are oxidized;one or more tryptophans are converted to N-formylkynurenine; one or moretryptophans are mono-hydroxyl tryptophan; one or more tryptophans aredi-hydroxyl tryptophan; one or more tryptophans are tri-hydroxyltryptophan; one or more arginines are converted to Arg 3-deoxyglucosone;the C-terminal glycine is not present; and/or there are one or morenon-glycosylated glycosites.

Such compositions can be obtained from a clarified harvest made usingCDM comprising the anti-VEGF protein as well as its variants subjectedto, for example, a capture chromatography procedure. The capture step isan affinity chromatography procedure using, for example, a Protein Acolumn. When a sample extracted from the affinity step is analyzedusing, for example, liquid chromatography-mass spectrometry (LC-MS), oneor more of these variants may be detected.

In one exemplary embodiment, the compositions of the invention cancomprise an anti-VEGF protein sharing structural characteristics ofaflibercept which can be oxidized at one or more of the following:His86, His110, His145, His209, His95, His19 and/or His203 (or equivalentresidue positions on proteins sharing certain structural characteristicsof aflibercept); Trp58 and/or Trp138 (or equivalent residue positions onproteins sharing certain structural characteristics of aflibercept);Tyr64 (or equivalent positions on proteins sharing certain structuralcharacteristics of aflibercept); Phe44 and/or Phe166 (or equivalentresidue positions on proteins sharing certain structural characteristicsof aflibercept); and/or Met10, Met 20, Met163 and/or Met192 (orequivalent residue positions on proteins sharing certain structuralcharacteristics of aflibercept). Such compositions can be obtained froma clarified harvest made using CDM comprising aflibercept as well as itsoxo-variants subjected to a capture chromatography procedure. Thecapture step can be an affinity chromatography procedure using, forexample, a Protein A column. When a sample extracted from the affinitystep is analyzed using, for example, liquid chromatography-massspectrometry (LC-MS), one or more of these variants may be detected.

In one embodiment, the compositions of the invention can comprise a VEGFMiniTrap having the amino acid sequence of SEQ ID NO.: 46, which can beoxidized at His86, His110, His145, His209, His95, His19 and/or His203;Trp58 and/or Trp138; Tyr64; Phe44 and/or Phe166; and/or Met10, Met 20,Met163 and/or Met192. Such compositions can be obtained from theclarified harvest made using CDM comprising the VEGF MiniTrap as well asits oxo-variants subjected to a capture chromatography procedure. Thecapture step is an affinity chromatography procedure using, for example,a Protein A column—when analyzed using liquid chromatography-massspectrometry (LC-MS), one or more of these variants may be detected.

In some exemplary embodiments, compositions of the present invention cancomprise an anti-VEGF protein and its variants (including oxo-variants),wherein the amount of the protein variants in the composition can be atmost about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%,1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more of thepreceding. Such compositions can be obtained from the clarified harvestmade using CDM comprising the anti-VEGF protein as well as its variantssubjected to a capture chromatography procedure. The capture step is anaffinity chromatography procedure using, for example, a Protein Acolumn—when analyzed using liquid chromatography-mass spectrometry(LC-MS), one or more of these variants may be detected. In one aspect,the color of such a composition is no darker/more intense than, forexample, the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4,BY4-BY5 or BY5-BY6 and/or having a b* value characterized by CIE L*, a*,b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the compositioncomprises about 5 g/L or about 10 g/L of the anti-VEGF protein.

In other exemplary embodiments, compositions of the present inventioncan comprise an anti-VEGF protein and its variants, wherein the amountof the protein variants in the composition can be about 0% to about 20%,for example, about 0% to about 20%, about 0.05% to about 20%, about 0.1%to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%,about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about20%, about 3% to about 20%, about 4% to about 20%, about 5% to about20%, about 6% to about 20%, about 7% to about 20%, about 8% to about20%, about 9% to about 20%, about 10% to about 20%, about 0% to about10% , about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% toabout 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5%to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about1.5% to about 10%, about 2% to about 10%, about 3% to about 10%, about4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7%to about 10%, about 8% to about 10%, about 9% to about 10%, about 0% toabout 7.5% , about 0.05% to about 7.5%, about 0.1% to about 7.5%, about0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%,about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% toabout 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3%to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6%to about 7.5%, about 7% to about 7.5%, about 0% to about 5% , about0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2%to about 5%, about 3% to about 5%, about 4% to about 5% and rangeswithin one or more of the preceding. Such compositions can be obtainedperforming capture chromatography on a harvest sample. The capture stepis an affinity chromatography procedure using, for example, a Protein Acolumn. When a sample is analyzed using liquid chromatography-massspectrometry (LC-MS), one or more of these variants may be detected. Inone aspect, the color of such a composition is no darker/more intensethan, for example, the European Brown-Yellow Color Standard BY2-BY3,BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized byCIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein thecomposition comprises about 5 g/L or about 10 g/L of the anti-VEGFprotein.

In one embodiment, compositions of the present invention can comprise ananti-VEGF protein including its acidic species, wherein the amount ofthe acidic species in the composition can be about 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%,3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and rangeswithin one or more of the preceding. As discussed supra, such acidicspecies can be detected by various methods such as ion exchange, forexample, WCX (WCX-10 HPLC, a weak cation exchange chromatography), orIEF (isoelectric focusing). Commonly, acidic species elute earlier thanthe main peak during CEX or later than the main peak during AEX analysis(see FIG. 16 and FIG. 17). Compositions comprising acidic species can beobtained from biological material such as harvest or affinity producedmaterial using ion exchange chromatography.

In one aspect, the color of such a composition is no darker/more intensethan, for example, the European Brown-Yellow Color Standard BY2-BY3,BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized byCIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein thecomposition comprises about 5 g/L or about 10 g/L. As an example,referring to FIG. 16 and FIG. 17, fractions F1 and F2 represent acidicfractions which comprise the majority of the acidic species. Peaks 1 and2 of MT1 in FIG. 17 comprise the acidic species and fractions F1 and F2comprise the majority of the acidic fractions. The fractions comprisingsuch acidic species (F1 and F2) also showed a yellow-brown colorcompared to other fractions (FIG. 18B and FIG. 18C).

In another embodiment, compositions of the instant invention comprise ananti-VEGF protein including its acidic species, wherein the amount ofacidic species in the composition can be about 0% to about 20%, forexample, about 0% to about 20%, about 0.05% to about 20%, about 0.1% toabout 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4%to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%,about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%,about 3% to about 20%, about 4% to about 20%, about 5% to about 20%,about 6% to about 20%, about 7% to about 20%, about 8% to about 20%,about 9% to about 20%, about 10% to about 20%, about 0% to about 10% ,about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% toabout 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8%to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5%to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% toabout 10%, about 5% to about 10%, about 6% to about 10%, about 7% toabout 10%, about 8% to about 10%, about 9% to about 10%, about 0% toabout 7.5% , about 0.05% to about 7.5%, about 0.1% to about 7.5%, about0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%,about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% toabout 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3%to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6%to about 7.5%, about 7% to about 7.5%, about 0% to about 5% , about0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2%to about 5%, about 3% to about 5%, about 4% to about 5% and rangeswithin one or more of the preceding. As discussed above, such acidicspecies can be detected by various methods, such as ion exchange, forexample, WCX (WCX-10 HPLC, a weak cation exchange chromatography), orIEF (isoelectric focusing). Typically, acidic species elute earlier thanthe main peak during CEX or later than the main peak during AEX analysis(See FIG. 16 and FIG. 17).

Using a cation exchange column, all peaks eluting prior to the main peakof interest were summed as the acidic region, and all peaks elutingafter the protein of interest were summed as the basic region. Inexemplary embodiments, the acidic species can be eluted as two or moreacidic regions and can be numbered AR1, AR2, AR3 and so on based on acertain retention time of the peaks and on the ion exchange column used.

In one embodiment, compositions can comprise an anti-VEGF proteinincluding acidic species, wherein AR1 is 20%, 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%,1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of thepreceding. In one aspect, compositions can comprise an anti-VEGF proteinincluding its acidic species, wherein AR1 is about 0.0% to about 10%,about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%,about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%,about 8% to about 10%, or about 10% to about 15%, and ranges within oneor more of the preceding. As discussed above, such acidic regions can bedetected by various methods, such as ion exchange, for example, WCX(WCX-10 HPLC, a weak cation exchange chromatography), or IEF(isoelectric focusing). Commonly, acidic species elute earlier than themain peak during CEX or later than the main peak during AEX analysis(See FIG. 16 and FIG. 17).

In another embodiment, compositions can comprise an anti-VEGF proteinincluding acidic species, wherein AR2 is 20%, 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%,1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of thepreceding. In one aspect, compositions can comprise an anti-VEGF proteinincluding acidic species, wherein AR2 is about 0.0% to about 10%, about0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, about 8%to about 10%, or about 10% to about 15%, and ranges within one or moreof the preceding.

In one embodiment, compositions can comprise an anti-VEGF proteinincluding basic species, wherein the amount of the basic species in thecomposition can be at most about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%,1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%,0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one ormore of the preceding. In one aspect, compositions can comprise ananti-VEGF protein and its basic species, wherein the amount of the basicspecies in the composition compared to the anti-VEGF protein can be 0%to about 20%, e.g., about 0% to about 20%, about 0.05% to about 20%,about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% toabout 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9%to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2%to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% toabout 20%, about 6% to about 20%, about 7% to about 20%, about 8% toabout 20%, about 9% to about 20%, about 10% to about 20%, about 0% toabout 10% , about 0.05% to about 10%, about 0.1% to about 10%, about0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%,about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about10%, about 1.5% to about 10%, about 2% to about 10%, about 3% to about10%, about 4% to about 10%, about 5% to about 10%, about 6% to about10%, about 7% to about 10%, about 8% to about 10%, about 9% to about10%, about 0% to about 7.5% , about 0.05% to about 7.5%, about 0.1% toabout 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about0.4% to about 7.5%, about 0.5% to about 7.5%, about 0.6% to about 7.5%,about 0.7% to about 7.5%, about 0.8% to about 7.5%, about 0.9% to about7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% toabout 7.5%, about 3% to about 7.5%, about 4% to about 7.5%, about 5% toabout 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, about 0% toabout 5% , about 0.05% to about 5%, about 0.1% to about 5%, about 0.2%to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5%to about 5%, about 0.6% to about 5%, about 0.7% to about 5%, about 0.8%to about 5%, about 0.9% to about 5%, about 1% to about 5%, about 1.5% toabout 5%, about 2% to about 5%, about 3% to about 5%, about 4% to about5% and ranges within one or more of the preceding.

The basic species can be eluted as two or more basic regions and can benumbered BR1, BR2, BR3 and so on based on a certain retention time ofthe peaks and ion exchange used.

In one embodiment, compositions can comprise an anti-VEGF proteinincluding its basic species, wherein BR1 is 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%,1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of thepreceding. In one aspect, compositions can comprise an anti-VEGF proteinand its basic species, wherein BR1 is about 0.0% to about 10% , about0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, about 8%to about 10%, or about 10% to about 15%, and ranges within one or moreof the preceding.

In another embodiment, the composition can comprise an anti-VEGF proteinand its basic species, wherein BR2 is 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%,1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of thepreceding. In one aspect, compositions can comprise an anti-VEGF proteinand its basic species of the anti-VEGF protein, wherein BR2 is about0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5%to about 8%, about 8% to about 10%, or about 10% to about 15%, andranges within one or more of the preceding.

In another embodiment, the composition can comprise an anti-VEGF proteinand its basic species, wherein BR3 is 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%,1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of thepreceding. In one aspect, compositions can comprise an anti-VEGF proteinand its basic species of the anti-VEGF protein, wherein BR3 is about0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5%to about 8%, about 8% to about 10%, or about 10% to about 15%, andranges within one or more of the preceding.

Photo-Induced Oxidation of Aflibercept

In addition to discovering the different color characteristics orvariants of the anti-VEGF protein compositions produced using CDM, theinventors also discovered that such compositions can be artificiallyproduced in the laboratory by exposure to light.

Modified, including oxidized, variants of an anti-VEGF composition canbe produced by exposing an anti-VEGF protein to cool-white light orultraviolet light. In one aspect, the anti-VEGF composition can compriseabout 1.5 to about 50-fold increase in one or more modifiedoligopeptides, compared to the sample, wherein the oligopeptides areselected from the group consisting of:

-   DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID    NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19),    TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21),    SDTGRPFVEMYSEIPEIIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.:    23), SDTGRPFVEM*YSEIPEIIHMTEGR (SEQ ID NO.: 64),    SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.:    66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR (SEQ ID NO.: 28),    RIIW*DSR (SEQ ID NO.: 115), IIW*DSRK (SEQ ID NO.: 114),    TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69),    KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32),    wherein H* is a histidine is oxidized to 2-oxo-histidine, wherein C*    is a cysteine is carboxymethylated, wherein M* is a oxidized    methionine, wherein W* is a oxidized tryptophan, wherein Y* is a    oxidized tyrosine, and wherein F* is a oxidized phenylalanine. In a    further aspect, the anti-VEGF composition can comprise about 1.5 to    about 10-fold increase in one or more modified oligopeptides by    exposing an anti-VEGF composition to cool-white light for a period    of time, for example, about 30 hours. In another aspect, the    anti-VEGF composition can comprise about 1.5 to about 10-fold    increase in one or more modified oligopeptides by exposing a sample    to cool-white light for about 75 hours. In yet another aspect, the    anti-VEGF composition can comprise about 1.5 to about 20-fold    increase in one or more oligopeptides by exposing the sample to    cool-white light for about 100 hours. In yet another aspect, the    anti-VEGF composition can comprise about 1.5 to about 20-fold    increase in one or more oligopeptides by exposing the sample to    cool-white light for about 150 hours. In still another aspect, the    anti-VEGF composition can comprise about 1.5 to about 50-fold    increase in one or more oligopeptides by exposing the sample to    cool-white light for about 300 hours—see Example 4 below.

The anti-VEGF composition can comprise about 1.5 to about 3-foldincrease in one or more oligopeptides, as described above, by exposing asample of an anti-VEGF composition to ultraviolet light for about 4hours. In another aspect, the anti-VEGF composition can comprise about1.5 to about 10-fold increase in one or more oligopeptides by exposingthe sample to ultraviolet light for about 10 hours. In yet anotheraspect, the anti-VEGF composition can comprise about 1.5 to about10-fold increase in one or more oligopeptides by exposing the sample toultraviolet light for about 16 hours. In yet another aspect, theanti-VEGF composition can comprise about 1.5 to about 25-fold increasein one or more oligopeptides by exposing the sample to ultraviolet lightfor about 20 hours. In yet another aspect, the anti-VEGF composition cancomprise about 1.5 to about 25-fold increase in one or moreoligopeptides by exposing the sample matrix to ultraviolet light forabout 40 hours. See Example 4.

Glycodiversity—Anti-VEGF Protein Produced Using CDM

The compositions of this invention comprise an anti-VEGF protein,wherein the anti-VEGF protein produced in CDM has a variety ofglycodiversity. The different glycosylation profiles of the anti-VEGFprotein are within the scope of this invention.

In some exemplary embodiments of the invention, the composition cancomprise an anti-VEGF protein glycosylated at one or more asparagines asfollows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAcglycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation;G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FSglycosylation; G2F glycosylation; G2FS glycosylation; G2FS2glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2G1cNAcglycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1glycosylation; Man6 glycosylation; Man6_G0+Phosphate glycosylation;Man6+Phosphate glycosylation; and/or Man7 glycosylation. In one aspect,the protein of interest can be aflibercept, anti-VEGF antibody or VEGFMiniTrap.

In one embodiment, the composition can have a glycosylation profile asfollows: about 40% to about 50% total fucosylated glycans, about 30% toabout 50% total sialylated glycans, about 6% to about 15% mannose-5, andabout 60% to about 79% galactosylated glycans. (Example 6).

In one embodiment, the composition can comprise an anti-VEGF protein,wherein the protein of interest has Man5 glycosylation at about 32.4% ofasparagine 123 residues and/or about 27.1% of asparagine 196 residues.In one aspect, the protein of interest can be aflibercept, anti-VEGFantibody or VEGF MiniTrap.

In another embodiment, the composition can have about 40%, about 41%,about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about48%, about 49% or about 50% total fucosylated glycans.

In yet another embodiment, the composition can have about 30%, about31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about44%, about 45%, about 46%, about 47%, about 48%, about 49% or about 50%total sialylated glycans.

In one embodiment, the composition can have about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, orabout 15% mannose-5.

In another embodiment, the composition can have about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, or about 79% totalgalactosylated glycans.

In one embodiment, the anti-VEGF protein can have a decreased level offucosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. The anti-VEGFprotein can have a decreased level of fucosylated glycans by rangeswithin one or more of the preceding values, for example, 1-10%, 1-15%,1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%,1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%,2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%,2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%,3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%,4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%,4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the levelof fucosylated glycans in an anti-VEGF protein produced using a soyhydrolysate.

In one embodiment, the anti-VEGF protein can have a decreased level ofsialylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. The anti-VEGFprotein can have a decreased level of sialylated glycans by rangeswithin one or more of the preceding values, for example, 1-10%, 1-15%,1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%,1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%,2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%,2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%,3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%,4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%,4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the levelof sialylated glycans in an anti-VEGF protein produced using a soyhydrolysate.

In another embodiment, the anti-VEGF protein can have a decreased levelof galactosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%,3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. The anti-VEGFprotein can have a decreased level of galactosylated glycans by rangeswithin one or more of the preceding values, for example, 1-10%, 1-15%,1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%,1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%,2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%,2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%,3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%,4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%,4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the levelof galactosylated glycans in an anti-VEGF protein produced using a soyhydrolysate.

In one embodiment, the anti-VEGF protein can have an increased level ofmannosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. The anti-VEGFprotein can have an increased level of mannosylated glycans by rangeswithin one or more of the preceding values, for example, 1-10%, 1-15%,1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%,1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%,2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%,2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%,3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%,4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%,4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the levelof mannosylated glycans in an anti-VEGF protein produced using a soyhydrolysate.

The compositions described in this section can be produced by severalupstream and downstream parameters as described below in sections IV andV, respectively.

IV. Preparation of Compositions Using Upstream Process Technologies

For biologics, the implementation of a robust and flexible upstreamprocess is desirable. An efficient upstream process can lead todesirable production and scale-up of a protein of interest. Theinventors discovered that the compositions of the invention comprisingan anti-VEGF protein can be produced by modulating conditions duringupstream protein production, such as changes in media components of aCDM. Each step in an upstream process may affect quality, purity andquantity of the manufactured protein.

The present disclosure provides evidence for the existence of certainvariants of aflibercept and/or MiniTrap produced using CDM. Thesevariants include isoforms that comprise one or more oxidized amino acidresidues. Examples of oxidized residues include, but are not limited to,one or more histidine, tryptophan, methionine, phenylalanine or tyrosineresidues. The compositions produced by using the modified CDM canproduce a preparation of anti-VEGF protein with a desired target valueof protein variants of aflibercept and/or MiniTrap. As alluded to above,there can also be a yellow-brownish color associated with fractionsproduced using a CDM. (As mentioned above, not all CDMs tested by theinventors manifested a distinct discoloration.)

This invention includes culturing a host cell in a modified CDM undersuitable conditions in which the cell expresses a recombinant protein ofinterest followed by harvesting a preparation of the recombinant proteinof interest produced by the cell. Such a modified CDM can be used toproduce the compositions as described above in Section III.

In one embodiment, the method comprises culturing a host cell in a CDMunder suitable conditions, wherein the host cell expresses a recombinantprotein of interest, such as aflibercept. The method further comprisesharvesting a preparation of the recombinant protein of interest producedby the cell, wherein the suitable conditions include a CDM with a:cumulative concentration of iron in said CDM that is less than about 55μM, cumulative concentration of copper in said CDM that is less than orequal to about 0.8 μM, cumulative concentration of nickel in said CDMthat is less than or equal to about 0.40 μM, cumulative concentration ofzinc in said CDM that is less than or equal to about 56 μM, cumulativeconcentration of cysteine in said CDM that is less than about 10 mM;and/or an antioxidant in said CDM in a concentration of about 0.001 mMto about 10 mM for a single antioxidant and no more than about 30 mMcumulative concentration if multiple antioxidants are added in said CDM.

In one aspect of the present embodiment, the preparation obtained fromusing suitable conditions results in a reduction in protein variants ofaflibercept and VEGF MiniTrap to a desired amount of protein variants ofaflibercept and VEGF MiniTrap (referred to as a “target value” ofprotein variants of aflibercept and VEGF MiniTrap). In a further aspectof this embodiment, the preparation obtained from using suitableconditions results in a reduction in color of the preparations to adesired BY value (referred to as a “target BY value”) when thepreparation of protein, including variants of aflibercept and VEGFMiniTrap, are normalized to a concentration of 5 g/L, 10 g/L or evenhigher.

In a further aspect of the present embodiment, the target BY valueand/or target value of variants can be obtained in a preparation wherethe titer increases or does not significantly decrease (see Example 5).

In some embodiments, the compositions produced by using the modified CDMcan produce a preparation of anti-VEGF protein with a desired target BYvalue, wherein the color of the preparation is characterized as follows:

-   (i) no more yellow-brown than European Color Standard BY2;-   (ii) no more yellow-brown than European Color Standard BY3;-   (iii) no more yellow-brown than European Color Standard BY4;-   (iv) no more yellow-brown than European Color Standard BY5;-   (v) between European Color Standard BY2 and BY3;-   (vi) between European Color Standard BY3 and BY4;-   (vii) between European Color Standard BY4 and BY5, wherein the    composition comprises about 5 g/L or about 10 g/L of the anti-VEGF    protein and wherein a sample of the composition can be obtained as a    sample from a Protein A eluate of a clarified harvest. As seen in    Example 9, Table 9-3 below, the Protein A eluate comprising 5 g/L    aflibercept exhibited a yellow-brown color measured as having a b*    value of 1.77. Such a sample when produced downstream following AEX    had a b* value of 0.50 demonstrating the utility of AEX to lower the    yellow-brown coloration of a sample (Table 9-3).

The compositions produced by using the modified CDM can produce apreparation of anti-VEGF protein, wherein the color of the preparationis characterized by a recognized standard color characterization in theCIELAB scale:

-   (i) no more yellow-brown than a b* value of about 22-23;-   (ii) no more yellow-brown than a b* value of about 16-17;-   (iii) no more yellow-brown than a b* value of 9-10;-   (iv) no more yellow-brown than a b* value of 4-5;-   (v) no more yellow-brown than a b* value of 2-3;-   (vi) between b* value of 17-23;-   (vii) between b* value of 10-17;-   (viii) between b* value of 5-10;-   (ix) between b* value of 3-5; or-   (x) between b* value of 1-3, wherein the composition comprises about    5 g/L or about 10 g/L of the anti-VEGF protein and wherein the    composition is obtained as a sample from a Protein A eluate of a    clarified harvest. See Example 9, Table 9-3.

For components added to the cell culture to form the modified CDM, theterm “cumulative amount” refers to the total amount of a particularcomponent added to a bioreactor over the course of the cell culture toform the CDM, including amounts added at the beginning of the culture(CDM at day 0) and subsequently added amounts of the component. Amountsof a component added to a seed-train culture or inoculum prior to thebioreactor production (i.e., prior to the CDM at day 0) are alsoincluded when calculating the cumulative amount of the component. Acumulative amount is unaffected by the loss of a component over timeduring the culture (for example, through metabolism or chemicaldegradation). Thus, two cultures with the same cumulative amounts of acomponent may nonetheless have different absolute levels, for example,if the component is added to the two cultures at different times (e.g.,if in one culture all of the component is added at the outset, and inanother culture the component is added over time). A cumulative amountis also unaffected by in situ synthesis of a component over time duringthe culture (for example, via metabolism or chemical conversion). Thus,two cultures with the same cumulative amounts of a given component maynonetheless have different absolute levels, for example, if thecomponent is synthesized in situ in one of the two cultures by way of abioconversion process. A cumulative amount may be expressed in unitssuch as, for example, grams or moles of the component. The term“cumulative concentration” refers to the cumulative amount of acomponent divided by the volume of liquid in the bioreactor at thebeginning of the production batch, including the contribution to thestarting volume from any inoculum used in the culture. For example, if abioreactor contains 2 liters of cell culture medium at the beginning ofthe production batch, and one gram of component X is added at days 0, 1,2, and 3, then the cumulative concentration after day 3 is 2 g/L (i.e.,4 grams divided by 2 liters). If, on day 4, an additional one liter ofliquid not containing component X were added to the bioreactor, thecumulative concentration would remain 2 g/L. If, on day 5, some quantityof liquid were lost from the bioreactor (for example, throughevaporation), the cumulative concentration would remain 2 g/L. Acumulative concentration may be expressed in units such as, for example,grams per liter or moles per liter.

A. Amino Acids:

In some embodiments, a modified CDM can be obtained by decreasing orincreasing cumulative concentrations of amino acids in a CDM.Non-limiting examples of such amino acids include alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine (or saltsthereof). The increase or decrease in the cumulative amount of theseamino acids in the modified CDM can be of about 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100% as compared to the starting CDM, and ranges within one or moreof the preceding. Alternatively, the increase or decrease in thecumulative amount of the one or more amino acids in the modified CDM canbe about 5% to about 20%, about 10% to about 30%, about 30% to about40%, about 30% to about 50%, about 40% to about 60%, about 60% to about70%, about 70% to about 80%, about 80% to about 90%, or about 90% toabout 100% as compared to the unmodified CDM, and ranges within one ormore of the preceding (see FIGS. 25-27 and Example 5).

In some embodiments, the modified CDM can be obtained by decreasing thecumulative concentration of cysteine in a CDM. The decrease in theamount of the cysteine in the CDM to form the modified CDM can be about1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% as compared to the unmodified CDM, andranges within one or more of the preceding. Alternatively, the decreasein the cumulative amount of the cysteine in the modified CDM can beabout 5% to about 20%, about 10% to about 30%, about 30% to about 40%,about 30% to about 50%, about 40% to about 60%, about 60% to about 70%,about 70% to about 80%, about 80% to about 90%, or about 90% to about100% as compared to the CDM, and ranges within one or more of thepreceding. In one aspect, the amount of cumulative cysteine in modifiedCDM is less than about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM or about 10 mM (seeFIGS. 25-27 and Example 5).

In some embodiments, the modified CDM can be obtained by replacing atleast a certain percentage of cumulative cysteine in a CDM with cystine.The replacement can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ascompared to the unmodified CDM, and ranges within one or more of thepreceding. Alternatively, the replacement can be about 5% to about 20%,about 10% to about 30%, about 30% to about 40%, about 30% to about 50%,about 40% to about 60%, about 60% to about 70%, about 70% to about 80%,about 80% to about 90%, or about 90% to about 100% as compared to theunmodified CDM, and ranges within one or more of the preceding (seeFIGS. 25-27 and Example 5).

In some embodiments, the modified CDM can be obtained by replacing atleast a certain percentage of cumulative cysteine in a CDM with cysteinesulfate. The replacement can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%as compared to the unmodified CDM, and ranges within one or more of thepreceding. Alternatively, the replacement can be about 5% to about 20%,about 10% to about 30%, about 30% to about 40%, about 30% to about 50%,about 40% to about 60%, about 60% to about 70%, about 70% to about 80%,about 80% to about 90%, or about 90% to about 100% as compared to theunmodified CDM, and ranges within one or more of the preceding.

B. Metals:

In some embodiments, the modified CDM can be obtained by decreasing orincreasing cumulative concentration of metals in a CDM. Non-limitingexamples of metals include iron, copper, manganese, molybdenum, zinc,nickel, calcium, potassium and sodium. The increase or decrease in theamount of the one or more metals in the modified CDM can be of about 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% as compared to the unmodified CDM, andranges within one or more of the preceding. Alternatively, the increaseor decrease in the cumulative amount of the one or more metals in themodified CDM can be about 5% to about 20%, about 10% to about 30%, about30% to about 40%, about 30% to about 50%, about 40% to about 60%, about60% to about 70%, about 70% to about 80%, about 80% to about 90%, orabout 90% to about 100% as compared to the unmodified CDM, and rangeswithin one or more of the preceding (see FIGS. 25-27 and Example 5).

C. Antioxidants:

In some embodiments, the modified CDM comprises one or moreantioxidants. Non-limiting examples of antioxidants can include taurine,hypotaurine, glycine, thioctic acid, glutathione, choline chloride,hydrocortisone, Vitamin C, Vitamin E and combinations thereof (see FIG.28A-E and Example 5).

In some embodiments, the modified CDM comprises about 0.01 mM to about20 mM of taurine, i.e., about 0.01 mM to about 1 mM, about 0.01 mM toabout 5 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM,about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM toabout 5 mM, about 1 mM to about 10 mM, and ranges within one or more ofthe preceding.

In some embodiments, the modified CDM comprises about 0.01 mM to about20 mM of hypotaurine, i.e., about 0.01 mM to about 1 mM, about 0.01 mMto about 5 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM,about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM toabout 5 mM, about 1 mM to about 10 mM, and ranges within one or more ofthe preceding.

In some embodiments, the modified CDM comprises about 0.01 mM to about20 mM of glycine, i.e., about 0.01 mM to about 1 mM, about 0.01 mM toabout 5 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM,about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM toabout 5 mM, about 1 mM to about 10 mM, and ranges within one or more ofthe preceding.

In some embodiments, the modified CDM comprises about 0.01 μM to about 5μM of thioctic acid, i.e., about 0.01 μM to about 0.1 μM, about 0.1 μMto about 1 μM, about 1 μM to about 2.5 μM, about 1 μM to about 3 μM,about 1 μM to about 5 μM, and ranges within one or more of thepreceding.

In some embodiments, the modified CDM comprises about 0.01 M to about 5mM of glutathione, i.e., about 0.01 mM to about 1 mM, about 0.1 mM toabout 1 mM, about 0.1 mM to about 5 mM, about 1 mM to about 5 mM, andranges within one or more of the preceding.

In some embodiments, the modified CDM comprises about 0.01 μM to about 5μM of hydrocortisone, i.e., about 0.01 μM to about 0.1 μM, about 0.1 μMto about 1 μM, about 1 μM to about 2.5 μM, about 1 μM to about 3 μM,about 1 μM to about 5 μM, and ranges within one or more of thepreceding.

In some embodiments, the modified CDM comprises about 1 μM to about 50μM of vitamin C, i.e., about 1 μM to about 5 μM, about 5 μM to about 20μM, about 10 μM to about 30 μM, about 5 μM to about 30 μM, about 20 μMto about 50 μM, about 25 μM to about 50 μM, and ranges within one ormore of the preceding.

D. Changes to the Media to Modulate Glycosylation:

This disclosure also includes methods of modulating glycosylation of ananti-VEGF protein by varying cumulative concentrations of certaincomponents in a CDM. Based on the cumulative amounts of components addedto the CDM, the total % fucosylation, total % galactosylation, total %sialylation and mannose-5 can be varied.

In exemplary embodiments, the method of modulating glycosylation of ananti-VEGF protein can comprise supplementing the CDM with uridine. Theanti-VEGF protein can have about 40% to about 50% total fucosylatedglycans, about 30% to about 55% total sialylated glycans, about 2% toabout 15% mannose-5, and about 60% to about 79% galactosylated glycans.(See Example 6 below).

In some embodiments, the method of modulating glycosylation of ananti-VEGF protein can comprise supplementing a CDM with manganese. Inone aspect, the CDM is devoid of manganese before supplementation. Theanti-VEGF protein can have about 40% to about 50% total fucosylatedglycans, about 30% to about 55% total sialylated glycans, about 2% toabout 15% mannose-5, and about 60% to about 79% galactosylated glycans.(See Example 6 below).

In some embodiments, the method of modulating glycosylation of ananti-VEGF protein can comprise supplementing a CDM with galactose. Inone aspect, the CDM is devoid of galactose before supplementation. Theanti-VEGF protein can have about 40% to about 50% total fucosylatedglycans, about 30% to about 55% total sialylated glycans, about 2% toabout 15% mannose-5, and about 60% to about 79% galactosylated glycans.(See Example 6 below).

In some embodiments, the method of modulating glycosylation of ananti-VEGF protein can comprise supplementing a CDM with dexamethasone.In one aspect, the CDM is devoid of dexamethasone beforesupplementation. The anti-VEGF protein can have about 40% to about 50%total fucosylated glycans, about 30% to about 55% total sialylatedglycans, about 2% to about 15% mannose-5, and about 60% to about 79%galactosylated glycans. (See Example 6 below).

In some embodiments, the method of modulating glycosylation of ananti-VEGF protein can comprise supplementing a CDM with one or more ofuridine, manganese, galactose and dexamethasone. In one aspect, the CDMis devoid of one or more of uridine, manganese, galactose anddexamethasone before supplementation. The anti-VEGF protein can haveabout 40% to about 50% total fucosylated glycans, about 30% to about 55%total sialylated glycans, about 2% to about 15% mannose-5, and about 60%to about 79% galactosylated glycans. (See Example 6 below).

V. Preparation of Compositions Using Downstream Process Technologies

The compositions comprising an anti-VEGF protein of the invention can beproduced by modulating conditions during downstream protein production.The inventors discovered that optimizing the downstream procedures canlead to minimization of certain variants of the anti-VEGF protein aswell as discoloration. Optimization of the downstream process mayproduce a composition with reduced oxo-variants as well as optimizedcolor characteristics.

The downstream process technologies may be used alone or in combinationwith the upstream process technologies described in Section IV, supra.

A. Anion-Exchange Chromatography:

In some embodiments, a composition of the invention can involve aprocess comprising: expressing an anti-VEGF protein in a host cell in aCDM, wherein the anti-VEGF protein is secreted from the host cell intothe medium and a clarified harvest is obtained. The harvest is subjectedto the following steps: (a) loading a biological sample obtained fromthe harvest onto an anion-exchange chromatography (AEX) column; (b)washing the AEX column with a suitable wash buffer, (c) collecting theflowthrough fraction(s), optionally, (d) washing the column with asuitable strip buffer and (e) collecting stripped fractions.

The flowthrough fractions can comprise oxo-variants of the anti-VEGFprotein which are about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anti-VEGFprotein sample when compared to the oxo-variants in the strippedfraction of the anion-exchange chromatography column. For example,referring to Table 9-5 and Table 9-6, the flowthrough fractions compriseoxidized variants of anti-VEGF protein where several histidine andtryptophan residues are about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% (and ranges withinone or more of the preceding) oxidized when compared against theoxidized variants in the stripped fractions.

The pH of both the equilibration and wash buffers for the AEX column canbe from about 8.20 to about 8.60. In another aspect, the conductivity ofboth the equilibration and wash buffers for the AEX column can be fromabout 1.50 to about 3.0 mS/cm. In one aspect, the equilibration and washbuffers can be about 50 mM Tris hydrochloride. In one aspect, the stripbuffer comprises 2 M sodium chloride or 1 N sodium hydroxide or both(see Table 2-2). Example 2 further illustrates optimizing theconcentration and conductivity of the equilibration and wash buffers.

Protein variants can include modifications of one or more residues asfollows: one or more asparagines are deamidated; one or more asparticacids are converted to iso-aspartate and/or Asn; one or more methioninesare oxidized; one or more tryptophans are converted toN-formylkynurenine; one or more tryptophans are mono-hydroxyltryptophan; one or more tryptophans are di-hydroxyl tryptophan; one ormore tryptophans are tri-hydroxyl tryptophan; one or more arginines areconverted to Arg 3-deoxyglucosone; the C-terminal glycine is notpresent; and/or there are one or more non-glycosylated glycosites.

The protein of interest can be aflibercept, anti-VEGF antibody or a VEGFMiniTrap. The protein variants can be formed by one or more of (i)oxidation of histidines from the histidine residues selected from His86,His110, His145, His209, His95, His19 and/or His203 (or equivalentresidue positions on proteins sharing certain structural characteristicsof aflibercept); (ii) oxidation of tryptophan residues selected fromtryptophan residues at Trp58 and/or Trp138 (or equivalent residuepositions on proteins sharing certain structural characteristics ofaflibercept); (iii) oxidation tyrosine residue at Tyr64 (or equivalentpositions on proteins sharing certain structural characteristics ofaflibercept); (iv) oxidation of phenylalanine residues selected fromPhe44 and/or Phe166 (or equivalent residue positions on proteins sharingcertain structural characteristics of aflibercept); and/or (v) oxidationof methionine residues selected from Met10, Met 20, Met163 and/or Met192(or equivalent residue positions on proteins sharing certain structuralcharacteristics of aflibercept).

The flowthrough fractions can comprise one or more of the following:

-   (a) a percentage of histidine residues which have been oxidized to    2-oxo-histidine wherein their color characterization is as follows:-   (i) no more yellow-brown than European Color Standard BY2;-   (ii) no more yellow-brown than European Color Standard BY3;-   (iii) no more yellow-brown than European Color Standard BY4;-   (iv) no more yellow-brown than European Color Standard BY5;-   (v) between European Color Standard BY2 and BY3;-   (vi) between European Color Standard BY3 and BY4;-   (vii) between European Color Standard BY4 and BY5, wherein the    composition comprises about 5 g/L or about 10 g/L of the anti-VEGF    protein, and wherein the composition is obtained as a sample from    the flowthrough fractions.-   (b) a percentage of histidine residues which have been oxidized to    2-oxo-histidine. Further, their color is characterized by having a    yellow-brown color which approximates that of BY2, BY3, BY4, BY5,    BY6, BY7; or is no darker/more intense than BY2, no darker than BY3,    no darker than BY4, no darker than BY5, no darker than BY6, no    darker than BY7; or is between that of BY2 and BY3, between that of    BY2 and BY4, between that of BY3 and BY4 or between that of BY3 and    BY5.-   (c) a percentage of histidine residues which have been oxidized to    2-oxo-histidine wherein their color is characterized by a color in    the CIE L*, a*, b* color space as follows:-   (i) no more yellow-brown than a b* value of about 22-23;-   (ii) no more yellow-brown than a b* value of about 16-17;-   (iii) no more yellow-brown than a b* value of 9-10;-   (iv) no more yellow-brown than a b* value of 4-5;-   (v) no more yellow-brown than a b* value of 2-3;-   (vi) between b* value of 17-23;-   (vii) between b* value of 10-17;-   (viii) between b* value of 5-10;-   (ix) between b* value of 3-5; or-   (x) between b* value of 1-3, wherein the composition comprises about    5 g/L or about 10 g/L of the anti-VEGF protein and wherein the    composition is obtained as a sample from the flowthrough fractions.-   (d) no more than about 1%, no more than about 0.1%, or about 0.1-1%,    0.2-1%, 0.3-1%, 0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1% or 0.9-1% of    histidine residues in the composition are oxidized to    2-oxo-histidine. The percentage calculation is described in Section    II.

B. Affinity Chromatography:

In some embodiments, compositions of the invention can be produced usinga process comprising: expressing an anti-VEGF protein in a host cellwherein anti-VEGF protein is secreted from the host cell into the mediumand a clarified harvest is obtained. The harvest is subjected to thefollowing steps, comprising (a) loading a biological sample obtainedfrom the clarified harvest onto an affinity chromatography column,wherein the affinity chromatography comprises a protein capable ofselectively or specifically binding to the anti-VEGF protein; (b)washing the affinity chromatography column with a suitable elutionbuffer, and (c) collecting the eluted fraction(s). For example, asexemplified in Table 7-1 and Table 7-7 through 7-10, using VEGF₁₆₅ asthe protein capable of selectively or specifically binding to theanti-VEGF protein and collecting the eluted fractions as per the methodabove led to a successful production of MT5 (an anti-VEGF protein),aflibercept and an anti-VEGF scFv fragment. Table 7-1 also disclosessuccessful production of MT5 using (i) mAb 1 (a mouse anti-VEGFR1 mAbhuman IgG1 where SEQ ID NO.: 73 is a heavy chain and SEQ ID NO.: 74 is alight chain); (ii) mAb2 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ IDNO.: 75 is a heavy chain and SEQ ID NO.: 76 is a light chain); (iii)mAb3 (a mouse anti-VEGF-R1 mAb mouse IgG1 where SEQ ID NO.: 77 is aheavy chain and SEQ ID NO.: 78 is a light chain) and (iv) mAb4 (a mouseanti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 79 is a heavy chain and SEQID NO.: 80 is a light chain) as different proteins capable ofselectively or specifically binding to MT5.

With respect to step (a) above, the biological sample to be loaded ontothe affinity column can come from a sample in which the clarifiedharvest can be subjected to chromatography prior to affinity including,but not limited to, ion exchange chromatography (either anion orcation). Other chromatographic procedures well known to the skilledartisan can also be employed prior to use of the affinity step. Theimportant point is that a biological sample comprising an anti-VEGFprotein can be subjected to affinity chromatography.

In some embodiments, compositions of the invention can be produced usinga process comprising: expressing a VEGF MiniTrap protein in a host cellwherein the VEGF MiniTrap is secreted from the host cell into the mediumand wherein the medium can be further processed forming a clarifiedharvest. This harvest can be further processed by known chromatographicprocedures yielding a biological sample comprising a VEGF MiniTrap. Thisbiological sample can be further processed by employing the followingsteps, comprising (a) loading the biological sample onto an affinitychromatography column, wherein the affinity chromatography comprises aprotein capable of selectively or specifically binding to or interactingwith the VEGF MiniTrap protein; (b) washing the affinity chromatographycolumn with a suitable elution buffer and (c) collecting the elutedfraction(s). Referring again to Table 7-1, disclosed in this Table is asuccessful production of MT5 (VEGF MiniTrap) using (i) VEGF₁₆₅; (ii)mAb1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 73 is a heavychain and SEQ ID NO.: 74 is a light chain); (iii) mAb2 (a mouseanti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain and SEQID NO.: 76 is a light chain); (iv) mAb3 (a mouse anti-VEGF-R1 mAb mouseIgG1 where SEQ ID NO.: 77 is a heavy chain and SEQ ID NO.: 78 is a lightchain) and (v) mAb4 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ IDNO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain) asdifferent proteins capable of selectively or specifically binding to ofinteracting with MT5.

In one embodiment, affinity chromatography can also be used to isolateother MiniTrap proteins. Following cleavage of an aflibercept, a samplecomprising the cleaved aflibercept can be subjected to affinitychromatography using a binder specific for the cleaved aflibercept. Inone aspect, the binder can be an antibody or portion thereof.

Cleaving of the aflibercept can be facilitated using proteolyticdigestion of aflibercept with, for example, IdeS protease (FabRICATOR)or a variant thereof to generate the VEGF MiniTrap. Cleaving of theaflibercept with IdeS protease or a variant thereof can produce amixture of products including a Fc fragment and the VEGF MiniTrap. TheVEGF MiniTrap can be further processed by using one or more of theproduction strategies described herein.

In some exemplary embodiments, a protein capable of selectively orspecifically binding (“binder”) to or interacting with an anti-VEGFprotein, such as aflibercept or MiniTrap, can originate from a human ora mouse.

The affinity production process can further comprise equilibrating anaffinity column using an equilibration buffer before loading thebiological sample. Exemplary equilibration buffers can be 20 mM sodiumphosphate, pH 6-8 (esp. 7.2), 10 mM sodium phosphate, 500 mM NaCl, pH6-8 (esp. 7.2), 50 mM Tris pH 7-8, DPBS pH 7.4.

The biological sample can be loaded using a suitable buffer, such as,DPBS.

This affinity production process can further comprise washing anaffinity column with one or more wash buffers. The column can be washedone or multiple times. Further, the washes can also be collected as washfractions. The pH of the wash buffer can be from about 7.0 to about8.60. In one aspect, the wash buffer can be DPBS. In another aspect, thewash buffer can be 20 mM sodium phosphate, pH 6-8 (esp. 7.2), 10 mMsodium phosphate, 500 mM NaCl, pH 6-8 (esp. 7.2), 50 mM Tris pH 7-8, orDPBS pH 7.4.

This affinity process can further comprise washing an affinity columnwith one or more suitable elution buffers and collecting the elutedfractions. The column can be washed one or multiple times. Non-limitingexamples of such a suitable elution buffer includes: ammonium acetate(pH of about 2.0 to about 3.0), acetic acid (pH of about 2.0 to about3.2), glycine-HCl (pH of about 2.0 to about 3.0), sodium citrate (pH ofabout 2.0 to about 3.0), citric acid (pH of about 2.0 to about 3.0),potassium isothiocyanate (pH of about 2.0 to about 3.0), or combinationsthereof.

In some aspects, the eluted fractions can be neutralized using aneutralizing buffer. An example of such a neutralizing buffer is Tris toTris-HCl (pH of about 7.0 to about 9.0).

C. IdeS Mutants:

The IdeS protease used for the cleavage of an Fc fusion protein such asaflibercept will rapidly lose enzymatic activity under basic pHconditions, which can limit its use during the manufacture of VEGFMiniTrap. Thus, variants have been developed to be more stable at basicpH, for example, in the presence of a strong base such as NaOH. Suchbasic conditions can be 0.05 N NaOH for 1 hr or 0.1 N NaOH for 0.5 hr.

In some embodiments, an IdeS mutant can have an amino acid sequencecomprising at least about 70% sequence identity over its full length tothe amino acid sequences set forth in the group consisting of SEQ IDNO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11,SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQID NO.: 16. In some aspects, the amino acid sequence has about 75%, 80%,85%, 90%, 95% or about 100% sequence identity over its full length tothe amino acid sequences mentioned directly above.

In some embodiments, an IdeS mutant can have an isolated nucleic acidmolecule encoding a polypeptide with an amino acid sequence comprisingat least 70% sequence identity over its full length to the amino acidsequences as set forth in the group consisting of SEQ ID NO.: 2, SEQ IDNO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.:12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16.In some aspects, the amino acid sequence has about 75%, 80%, 85%, 90%,95% or about 100% sequence identity over its full length to the aminoacid sequences mentioned directly above.

In some embodiments, the polypeptide can have an amino acid sequencecomprising at least 70% sequence identity over its full length to theamino acid sequences as set forth in the group consisting of SEQ ID NO.:2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ IDNO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11,SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQID NO.: 16 and can be expressed by a host cell with a suitable vectorcomprising nucleic acid coding for the identified peptides. In oneaspect, the nucleic acid molecule is operatively linked to an expressioncontrol sequence capable of directing its expression in a host cell. Inone aspect, the vector can be a plasmid. In some aspects, the amino acidsequence has about 75%, 80%, 85%, 90%, 95% or about 100% sequenceidentity over its full length to the amino acid sequences mentioneddirectly above. In some aspects, an isolated nucleic acid molecule canbe used to encode the polypeptide.

In some embodiments, an IdeS mutant can have an amino acid sequencecomprising a parental amino acid sequence defined by SEQ ID NO.: 1(IdeS) with an asparagine residue at position 87, 130, 182 and/or 274mutated to an amino acid other than asparagine. In one aspect, themutation can confer an increased chemical stability at alkalinepH-values compared to the parental amino acid sequence. In anotheraspect, the mutation can confer an increase in chemical stability by 50%at alkaline pH-values compared to the parental amino acid sequence. Inone aspect, the amino acid can be selected from aspartic acid, leucine,and arginine. In a particular aspect, the asparagine residue at position87 is mutated to an aspartic acid residue. In another particular aspect,the asparagine residue at position 130 is mutated to an arginineresidue. In yet another particular aspect, the asparagine residue atposition 182 is mutated to a leucine residue. In yet another particularaspect, the asparagine residue at position 274 is mutated to an asparticacid residue. In yet another particular aspect, the asparagine residuesat position 87 and 130 are mutated. In yet another particular aspect,the asparagine residues at position 87 and 182 are mutated. In yetanother particular aspect, the asparagine residues at position 87 and274 are mutated. In yet another particular aspect, the asparagineresidues at position 130 and 182 are mutated. In yet another particularaspect, the asparagine residues at position 130 and 274 are mutated. Inyet another particular aspect, the asparagine residues at position 182and 274 are mutated. In yet another particular aspect, the asparagineresidues at position 87, 130 and 182 are mutated. In yet anotherparticular aspect, the asparagine residues at position 87, 182 and 274are mutated. In yet another particular aspect, the asparagine residuesat position 130, 182 and 274 are mutated. In yet another particularaspect, the asparagine residues at position 87, 130, 182 and 274 aremutated. In some aspects, the amino acid sequence has about 75%, 80%,85%, 90%, 95% or about 100% sequence identity over its full length tothe amino acid sequences described above. In some aspects, an isolatednucleic acid molecule can be used to encode the polypeptide.

Those of ordinary skill in the art familiar with standard molecularbiology techniques can without undue burden prepare and use IdeS mutantsof the present invention. Standard techniques can be used forrecombinant DNA, oligonucleotide synthesis, tissue culture, andtransformation (e.g., electroporation, lipofection). See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, supra, which isincorporated herein by reference for any purpose. Enzymatic reactionsand production techniques can be performed according to manufacturer'sspecification or as described herein.

VI. Protein Production Generally

A variety of different production techniques, including, but not limitedto, affinity, ion exchange, mixed mode, size exclusion, and hydrophobicinteraction chromatography, singularly or in combination, are envisagedto be within the scope of the present invention. These chromatographicsteps separate mixtures of proteins of a biological sample on the basisof their charge, degree of hydrophobicity, or size, or a combinationthereof, depending on the particular form of separation. Severaldifferent chromatography resins are available for each of the techniquesalluded to supra, allowing accurate tailoring of the production schemeto a particular protein involved. Each separation method results in theprotein traversing at different rates through a column to achieve aphysical separation that increases as they pass further through thecolumn or adhere selectively to a separation medium. The proteins arethen either (i) differentially eluted using an appropriate elutionbuffer and/or (ii) collected from flowthrough fractions obtained fromthe column used, optionally, from washing the column with an appropriateequilibration buffer. In some cases, the protein of interest isseparated from impurities (HCPs, protein variants, etc.) when theimpurities preferentially adhere to the column and the protein ofinterest less so, i.e., the protein of interest does not adsorb to thesolid phase of a particular column and thus flows through the column. Insome cases, the impurities are separated from the protein of interestwhen they fail to adsorb to the column and thus flow through the column.

The production process may begin at the separation step after therecombinant protein has been produced using upstream production methodsdescribed above and/or by alternative production methods conventional inthe art. Once a clarified solution or mixture comprising the protein ofinterest, for example, a fusion protein, has been obtained, separationof the protein of interest from process-related impurities (such as theother proteins produced by the cell (like HCPs), as well asproduct-related substances, such acidic or basic variants) is performed.A combination of one or more different production techniques, includingaffinity, ion exchange (e.g., CEX, AEX), mixed-mode (MM), and/orhydrophobic interaction chromatography can be employed. Such productionsteps separate mixtures of components within a biological sample on thebasis of their, for example, charge, degree of hydrophobicity, and/orapparent size. Numerous chromatography resins are commercially availablefor each of the chromatography techniques mentioned herein, allowingaccurate tailoring of the production scheme to a particular proteininvolved. Each of the separation methods allow proteins to eithertraverse at different rates through a column achieving a physicalseparation that increases as they pass further through the column or toadsorb selectively to a separation resin (or medium). The proteins canthen be differentially collected. In some cases, the protein of interestis separated from components of a biological sample when othercomponents specifically adsorb to a column's resin while the protein ofinterest does not.

A. Primary Recovery and Virus Inactivation

In certain embodiments, the initial steps of the production methodsdisclosed herein involve the clarification and primary recovery of aprotein of interest from a biological sample. The primary recovery willinclude one or more centrifugation steps to separate the protein ofinterest from a host cell and attendant cellular debris. Centrifugationof the sample can be performed at, for example, but not by way oflimitation, 7,000×g to approximately 12,750×g. In the context oflarge-scale production, such centrifugation can occur on-line with aflow rate set to achieve, for example, a turbidity level of 150 NTU inthe resulting supernatant. Such supernatant can then be collected forfurther processing or in-line filtered through one or more depth filtersfor further clarification of the sample.

In certain embodiments, the primary recovery may include the use of oneor more depth filtration steps to clarify the sample and, thereby, aidin processing the protein of interest. In other embodiments, the primaryrecovery may include the use of one or more depth filtration steps postcentrifugation. Non-limiting examples of depth filters that can be usedin the context of the instant invention include the Millistak+ X0HC,F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore), 3M™ model 30/60ZA,60/90 ZA, VR05, VR07, delipid depth filters (3M Corp.). A 0.2 μm filtersuch as Sartorius's 0.45/0.2 μm Sartopore™ bi-layer or Millipore'sExpress SHR or SHC filter cartridges typically follows the depthfilters. Other filters well known to the skilled artisan can also beused.

In certain embodiments, the primary recovery process can also be a pointto reduce or inactivate viruses that can be present in a biologicalsample. Any one or more of a variety of methods of viralreduction/inactivation can be used during the primary recovery phase ofproduction including heat inactivation (pasteurization), pHinactivation, buffer/detergent treatment, UV and γ-ray irradiation andthe addition of certain chemical inactivating agents such asβ-propiolactone or, for example, copper phenanthroline as described inU.S. Pat. No. 4,534,972, the entire teaching of which is incorporatedherein by reference. In certain exemplary embodiments of the presentinvention, the sample is exposed to detergent viral inactivation duringthe primary recovery phase. In other embodiments, the sample may beexposed to low pH inactivation during the primary recovery phase.

In those embodiments where viral reduction/inactivation is employed, abiological sample can be adjusted, as needed, for further productionsteps. For example, following low pH viral inactivation, the pH of thesample is typically adjusted to a more neutral pH, for example, fromabout 4.5 to about 8.5, prior to continuing the production process.Additionally, the mixture may be diluted with water for injection (WFI)to obtain a desired conductivity.

B. Affinity Chromatography

In certain exemplary embodiments, it may be advantageous to subject abiological sample to affinity chromatography for production of a proteinof interest. The chromatographic material is capable of selectively orspecifically binding to or interacting with the protein of interest.Non-limiting examples of such chromatographic material include: ProteinA and Protein G. Also included is chromatographic material comprising,for example, a protein or portion thereof capable of binding to orinteracting with the protein of interest. In one aspect, the protein ofinterest is an anti-VEGF protein such as aflibercept, MiniTrap or aprotein related thereto.

Affinity chromatography can involve subjecting a biological sample to acolumn comprising a suitable Protein A resin. When used herein, the term“Protein A” encompasses Protein A recovered from a native sourcethereof, Protein A produced synthetically (e.g., by peptide synthesis orby recombinant techniques), and variants thereof which retain theability to bind proteins which have a C_(H)2/C_(H)3 region. In certainaspects, Protein A resin is useful for affinity-based production andisolation of a variety of antibody isotypes by interacting specificallywith the Fc portion of a molecule should it possess that region.

There are several commercial sources for Protein A resin. One suitableresin is MabSelect™ from GE Healthcare. Suitable resins include, but arenot limited to, Mab Select SuRe™, Mab Select SuRe LX, Mab Select, MabSelect SuRe pcc, Mab Select Xtra, rProtein A Sepharose from GEHealthcare, ProSep HC, ProSep Ultra, and ProSep Ultra Plus from EMDMillipore, MapCapture from Life Technologies. A non-limiting example ofa suitable column packed with MabSelect™ is an about 1.0 cmdiameter×about 21.6 cm long column (17 mL bed volume). A suitable columnmay comprise a resin such as MabSelect™ SuRe or an analogous resin.Protein A can also be purchased commercially from Repligen, Pharmaciaand Fermatech.

An affinity column can be equilibrated with a suitable buffer prior tosample loading. Following loading of the column, the column can bewashed one or multiple times using a suitable wash buffer. The columncan then be eluted using an appropriate elution buffer, for example,glycine-HCl, acetic acid, or citric acid. The eluate can be monitoredusing techniques well known to those skilled in the art such as a UVdetector. The eluted fractions of interest can be collected and thenprepared for further processing.

In one aspect, the eluate may be subjected to viral inactivation, forexample, either by detergent or low pH. A suitable detergentconcentration or pH (and time) can be selected to obtain a desired viralinactivation result. After viral inactivation, the eluate is usually pHand/or conductivity adjusted for subsequent production steps.

The eluate may be subjected to filtration through a depth filter toremove turbidity and/or various impurities from the protein of interestprior to additional chromatographic polishing steps. Examples ofsuitable depth filters include, but are not limited to, Millistak+ XOHC,FOHC, DOHC, AIHC, X0SP, and BIHC Pod filters (EMD Millipore), or ZetaPlus 30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and VRO5 filters (3M). TheEmphaze AEX Hybrid Purifier multi-mechanism filter may also be used toclarify the eluate. The eluate pool may need to be adjusted to aparticular pH and conductivity in order to obtain desired impurityremoval and product recovery from the depth filtration step.

C. Anion Exchange Chromatography

In certain embodiments, a protein of interest is produced by subjectinga biological sample to at least one anion exchange separation step. Inone scenario, the anion exchange step can occur following an affinitychromatography procedure (e.g., Protein A affinity). In other scenarios,the anion exchange step can occur before the affinity chromatographystep. In yet other protocols, anion exchange can occur both before andafter an affinity chromatography step. In one aspect, the protein ofinterest is either aflibercept or MiniTrap.

The use of an anionic exchange material versus a cationic exchangematerial is based, in part, on the local charges of the protein ofinterest. Anion exchange chromatography can be used in combination withother chromatographic procedures such as affinity chromatography, sizeexclusion chromatography, hydrophobic interaction chromatography as wellas other modes of chromatography known to the skilled artisan.

In performing a separation, the initial protein composition (biologicalsample) can be placed in contact with an anion exchange material byusing any of a variety of techniques, for example, using a batchproduction technique or a chromatographic technique.

In the context of batch production, anion exchange material is preparedin, or equilibrated to, a desired starting buffer. Upon preparation, aslurry of the anion exchange material is obtained. The biological sampleis contacted with the slurry to allow for protein adsorption to theanion exchange material. A solution comprising acidic species that donot bind to the AEX material is separated from the slurry by allowingthe slurry to settle and removing the supernatant. The slurry can besubjected to one or more washing steps and/or elution steps.

In the context of chromatographic separation, a chromatographic columnis used to house chromatographic support material (resin or solidphase). A sample comprising a protein of interest is loaded onto aparticular chromatographic column. The column can then be subjected toone or more wash steps using a suitable wash buffer. Components of asample that have not adsorbed onto the resin will likely flow throughthe column. Components that have adsorbed to the resin can bedifferentially eluted using an appropriate elution buffer.

A wash step is typically performed in AEX chromatography usingconditions similar to the load conditions or alternatively by decreasingthe pH and/or increasing the ionic strength/conductivity of the wash ina step wise or linear gradient manner. In one aspect, the aqueous saltsolution used in both the loading and wash buffer has a pH that is at ornear the isoelectric point (p1) of the protein of interest. Typically,the pH is about 0 to 2 units higher or lower than the p1 of the proteinof interest, however it may be in the range of 0 to 0.5 units higher orlower. It may also be at the p1 of the protein of interest.

The anionic agent may be selected from the group consisting of acetate,chloride, formate and combinations thereof. The cationic agent may beselected from the group consisting of Tris, arginine, sodium andcombinations thereof. In a particular example, the buffer solution is aTris/formate buffer. The buffer may be selected from the groupconsisting of pyridine, piperazine, L-histidine, Bis-Tris, Bis-Trispropane, imidazole, N-ethylmorpholine, TEA (triethanolamine), Tris,morpholine, N-methyldiethanolamine, AMPD(2-amino-2-methyl-1,3-propanediol), diethanolamine, ethanolamine, AMP(2-amino-2-methyl-l-propaol), piperazine, 1,3-diaminopropane andpiperidine.

A packed anion-exchange chromatography column, anion-exchange membranedevice, anion-exchange monolithic device, or depth filter media can beoperated either in bind-elute mode, flowthrough mode, or a hybrid modewherein proteins exhibit binding to the chromatographic material and yetcan be washed from such material using a buffer that is the same orsubstantially similar to the loading buffer.

In the bind-elute mode, a column or membrane device is first conditionedwith a buffer with appropriate ionic strength and pH under conditionswhere certain proteins will adsorb to the resin-based matrix. Forexample, during the feed load, a protein of interest can be adsorbed tothe resin due to electrostatic attraction. After washing the column orthe membrane device with the equilibration buffer or another buffer witha different pH and/or conductivity, the product recovery is achieved byincreasing the ionic strength (i.e., conductivity) of the elution bufferto compete with the solute for the charged sites of the anion exchangematrix. Changing the pH and thereby altering the charge of the solute isanother way to achieve elution of the solute. The change in conductivityor pH may be gradual (gradient elution) or stepwise (step elution).

In the flowthrough mode, a column or membrane device is operated at aselected pH and conductivity such that the protein of interest does notbind to the resin or the membrane while the acidic species will eitherbe retained on the column or will have a distinct elution profile ascompared to the protein of interest. In the context of this strategy,acidic species will interact with or bind to the chromatographicmaterial under suitable conditions while the protein of interest andcertain aggregates and/or fragments of the protein of interest will flowthrough the column.

Non-limiting examples of anionic exchange resins includediethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternaryamine (Q) groups. Additional non-limiting examples include: Poros 50PIand Poros 50HQ, which are a rigid polymeric bead with a backboneconsisting of cross-linked poly[styrene-divinylbenzene]; Capto Q Impresand Capto DEAE, which are a high flow agarose bead; Toyopearl QAE-550,Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are a polymericbase bead; Fractogel® EMD TMAE Hicap, which is a synthetic polymericresin with a tentacle ion exchanger; Sartobind STIC® PA nano, which is asalt-tolerant chromatographic membrane with a primary amine ligand;Sartobind Q nano, which is a strong anion exchange chromatographicmembrane; CUNO BioCap, which is a zeta-plus depth filter mediaconstructed from inorganic filter aids, refined cellulose, and an ionexchange resin; and X0HC, which is a depth-filter media constructed frominorganic filter aid, cellulose, and mixed cellulose esters.

In certain embodiments, the protein load of a sample may be adjusted toa total protein load to the column of between about 50 g/L and about 500g/L, or between about 75 g/L and about 350 g/L, or between about 200 g/Land about 300 g/L. In other embodiments, the protein concentration ofthe load protein mixture is adjusted to a protein concentration of thematerial loaded to the column of about 0.5 g/L and about 50 g/L, betweenabout 1 g/L and about 20 g/L, or between about 3 g/L and about 10 g/L.In yet other embodiments, the protein concentration of the load proteinmixture is adjusted to a protein concentration of the material to thecolumn of about 37 g/L.

Additives such as polyethylene glycol (PEG), detergents, amino acids,sugars, chaotropic agents can be added to enhance the performance of theseparation to achieve better separation, recovery and/or productquality.

In certain embodiments, including those relating to aflibercept and/orVEGF MiniTrap, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove at least10% of protein variants, thereby producing protein compositions thathave reduced protein variants.

The protein variants can include modifications of one or more residuesas follows: one or more asparagines are deamidated; one or more asparticacids are converted to aspartate-glycine and/or Asn-Gly; one or moremethionines are oxidized; one or more tryptophans are converted toN-formylkynurenine; one or more tryptophans are mono-hydroxyltryptophan; one or more tryptophans are di-hydroxyl tryptophan; one ormore tryptophans are tri-hydroxyl tryptophan; one or more arginines areconverted to Arg 3-deoxyglucosone; the C-terminal glycine is notpresent; and/or there are one or more non-glycosylated glycosites. Theuse of AEX was also observed to reduce oxidized and acidic species ofanti-VEGF variants in said affinity eluate. Compared to the affinityeluate, following use of AEX, the flowthrough fraction may show areduction of at least about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,11%, 10%, 9%, 8%, 7%, 6%, or 5% in oxidized and/or acidic species ofanti-VEGF variants.

Protein variants of aflibercept and/or VEGF MiniTrap can include one ormore of (i) oxidated histidines from the histidine residues selectedfrom His86, His110, His145, His209, His95, His19 and/or His203; (ii)oxidated tryptophan residues selected from tryptophan residues at Trp58and/or Trp138; (iii) oxidated tyrosine residue at Tyr64; (iv) oxidatedphenylalanine residues selected from Phe44 and/or Phe166; and/or (v)oxidated methionine residues selected from Met10, Met 20, Met163 and/orMet192.

D. Cation Exchange Chromatography

The compositions of the present invention can be produced by subjectinga biological sample comprising a protein of interest to at least onecation exchange (CEX) step. In certain exemplary embodiments, the CEXstep will be in addition to an AEX step and occur either before or afterthe AEX step. In one aspect, the protein of interest is eitheraflibercept, MiniTrap or a molecule related thereto.

The use of a cationic exchange material versus an anionic exchangematerial, such as those anionic exchange materials discussed supra, isbased, in part, on the local charges of the protein of interest in agiven solution and the separation conditions desired. It is within thescope of this invention to employ a cationic exchange step prior to theuse of an anionic exchange step, or an anionic exchange step prior tothe use of a cationic exchange step. Furthermore, it is within the scopeof this invention to employ only a cationic exchange step in combinationwith other chromatography procedures.

In performing cation exchange, a sample comprising a protein of interestcan be contacted with a cation exchange material by using any of avariety of techniques, for example, using a batch production techniqueor a chromatographic technique, as described above for AEX.

An aqueous salt solution may be used as both a loading and wash bufferhaving a pH that is lower than the isoelectric point (pI) of the proteinof interest. In one aspect, the pH is about 0 to 5 units lower than thepI of the protein. In another aspect, it is in the range of 1 to 2 unitslower than the pI of the protein. In yet another aspect, it is in therange of 1 to 1.5 units lower than the pI of the protein.

In certain embodiments, the concentration of the anionic agent inaqueous salt solution is increased or decreased to achieve a pH ofbetween about 3.5 and about 10.5, or between about 4 and about 10, orbetween about 4.5 and about 9.5, or between about 5 and about 9, orbetween about 5.5 and about 8.5, or between about 6 and about 8, orbetween about 6.5 and about 7.5. In one aspect, the concentration ofanionic agent is increased or decreased in the aqueous salt solution inorder to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Buffersystems suitable for use in the CEX methods include, but are not limitedto, Tris formate, Tris acetate, ammonium sulfate, sodium chloride, andsodium sulfate.

In certain embodiments, the conductivity and pH of the aqueous saltsolution is adjusted by increasing or decreasing the concentration of acationic agent. In one aspect, the cationic agent is maintained at aconcentration ranging from about 20 mM to about 500 mM, about 50 mM toabout 350 mM, about 100 mM to about 300 mM, or about 100 mM to about 200mM. Non-limiting examples of the cationic agent can be selected from thegroup consisting of sodium, Tris, triethylamine, ammonium, arginine, andcombinations thereof.

A packed cation-exchange chromatography column or a cation-exchangemembrane device can be operated either in bind-elute mode, flowthroughmode, or a hybrid mode wherein the product exhibits binding to orinteracting with a chromatographic material yet can be washed from suchmaterial using a buffer that is the same or substantially similar to theloading buffer (details of these modes are outlined above).

Cationic substituents include carboxymethyl (CM), sulfoethyl (SE),sulfopropyl (SP), phosphate (P) and sulfonate (S). Additional cationicmaterials include, but are not limited to: Capto SP ImpRes, which is ahigh flow agarose bead; CM Hyper D grade F, which is a ceramic beadcoated and permeated with a functionalized hydrogel, 250-400 ionicgroups μeq/mL; Eshmuno S, which is a hydrophilic polyvinyl ether basematrix with 50-100 μeq/mL ionic capacity; Nuvia C Prime, which is ahydrophobic cation exchange media composed of a macroporous highlycrosslinked hydrophilic polymer matrix 55-75 με/mL; Nuvia S, which has aUNOsphere base matrix with 90-150 με/mL ionic groups; Poros HS, which isa rigid polymeric bead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene]; Poros XS, which is a rigid polymetic beadwith a backbone consisting of cross-linked poly[styrenedivinyl-benzene]; Toyo Pearl Giga Cap CM 650M, which is a polymeric basebead with 0.225 meq/mL ionic capacity; Toyo Pearl Giga Cap S 650M, whichis a polymeric base bead; and Toyo Pearl MX TRP, which is a polymericbase bead. It is noted that CEX chromatography can be used with MMresins, described herein.

The protein load of a sample comprising a protein of interest isadjusted to a total protein load to the column of between about 5 g/Land about 150 g/L, or between about 10 g/L and about 100 g/L, betweenabout 20 g/L and about 80 g/L, between about 30 g/L and about 50 g/L, orbetween about 40 g/L and about 50 g/L. In certain embodiments, theprotein concentration of the load protein mixture is adjusted to aprotein concentration of the material to be loaded onto the column ofabout 0.5 g/L and about 50 g/L, or between about 1 g/L and about 20 g/L.

Additives such as polyethylene glycol, detergents, amino acids, sugars,chaotropic agents can be added to enhance the performance of theseparation so as to achieve better separation, recovery and/or productquality.

In certain embodiments, including those relating to aflibercept oranti-VEGF antibody or VEGF MiniTrap, the methods of the instantinvention can be used to selectively remove, significantly reduce, oressentially remove all of the oxo-variants in a sample where the proteinof interest will essentially be in the flowthrough of a CEX procedurewhile the oxo-variants will be substantially captured by the columnmedia.

E. Mixed Mode Chromatography

Mixed mode (“MM”) chromatography may also be used to prepare thecompositions of the invention. MM chromatography, also referred toherein as “multimodal chromatography”, is a chromatographic strategythat utilizes a support comprising a ligand that is capable of providingat least two different interactions with an analyte or protein ofinterest from a sample. One of these sites provides an attractive typeof charge-charge interaction between the ligand and the protein ofinterest and the other site provides for electron acceptor-donorinteraction and/or hydrophobic and/or hydrophilic interactions. Electrondonor-acceptor interactions include interactions such ashydrogen-bonding, π-π, cation-π, charge transfer, dipole-dipole, induceddipole, etc.

The column resin employed for a mixed mode separation can be CaptoAdhere. Capto Adhere is a strong anion exchanger with multimodalfunctionality. Its base matrix is a highly cross-linked agarose with aligand (N-benzyl-N-methyl ethanol amine) that exhibits differentfunctionalities for interaction, such as ionic interaction, hydrogenbonding and hydrophobic interaction. In certain aspects, the resinemployed for a mixed mode separation is selected from PPA-HyperCel andHEA-HyperCel. The base matrices of PPA-HyperCel and HEA-HyperCel arehigh porosity cross-linked cellulose. Their ligands arephenylpropylamine and hexylamine, respectively. Phenylpropylamine andhexylamine offer different selectivity and hydrophobicity options forprotein separations. Additional mixed mode chromatographic supportsinclude, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M,and Eshmuno® HCX. In certain aspects, the mixed mode chromatographyresin is comprised of ligands coupled to an organic or inorganicsupport, sometimes denoted by a base matrix, directly or via a spacer.The support may be in the form of particles, such as essentiallyspherical particles, a monolith, filter, membrane, surface, capillaries,and the like. In certain aspects, the support is prepared from a nativepolymer, such as cross-linked carbohydrate material, such as agarose,agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan,alginate and the like. To obtain high adsorption capacities, the supportcan be porous, and ligands are then coupled to the external surfaces aswell as to the pore surfaces. Such native polymer supports can beprepared according to standard methods, such as inverse suspensiongelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), theentire teaching of which is incorporated herein by reference).Alternatively, the support can be prepared from a synthetic polymer,such as cross-linked synthetic polymers, for example, styrene or styrenederivatives, divinylbenzene, acrylamides, acrylate esters, methacrylateesters, vinyl esters, vinyl amides, and the like. Such syntheticpolymers can be produced according to standard methods, see “Styrenebased polymer supports developed by suspension polymerization” (RArshady: Chimica e L'Industria 70(9), 70-75 (1988), the entire teachingof which is incorporated herein by reference). Porous native orsynthetic polymer supports are also available from commercial sources,such as GE Healthcare, Uppsala, Sweden.

The protein load of a biological sample mixture comprising a protein ofinterest can be adjusted to a total protein load to the column ofbetween about 25 g/L and about 750 g/L, or between about 75 g/L andabout 500 g/L, or between about 100 g/L and about 300 g/L. In certainexemplary embodiments, the protein concentration of the load proteinmixture is adjusted to a protein concentration of the material loaded tothe column of about 1 g/L and about 50 g/L, or between about 9 g/L andabout 25 g/L.

Additives such as polyethylene glycol, detergents, amino acids, sugars,chaotropic agents can be added to enhance the performance of theseparation, so as to achieve better separation, recovery and/or productquality.

In certain embodiments, including those relating to aflibercept and/orMiniTrap, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe PTMs, including oxo-variants.

The methods for producing the composition of the invention can also beimplemented in a continuous chromatography mode. In this mode, at leasttwo columns are employed (referred to as a “first” column and a “second”column). In certain embodiments, this continuous chromatography mode canbe performed such that the eluted fractions and/or stripped fractionscomprising PTMs, for example, oxo-variants, can then be loadedsubsequently or concurrently onto the second column (with or withoutdilution).

In one embodiment, the media choice for continuous mode can be one ofmany chromatographic resins with pendant hydrophobic and anion exchangefunctional groups, monolithic media, membrane adsorbent media or depthfiltration media.

F. Hydrophobic Interaction Chromatography

The compositions of the invention may also be prepared using hydrophobicinteraction chromatography (HIC).

In performing the separation, a biological sample is contacted with aHIC material, for example, using a batch production technique or using acolumn or membrane chromatography. Prior to HIC processing it may bedesirable to adjust the concentration of the salt buffer to achievedesired protein binding/interaction to the resin or the membrane.

Whereas ion exchange chromatography relies on the local charge of theprotein of interest for selective separation, hydrophobic interactionchromatography exploits the hydrophobic properties of proteins toachieve selective separation. Hydrophobic groups on or within a proteininteract with hydrophobic groups of chromatography resin or a membrane.Typically, under suitable conditions, the more hydrophobic a protein is(or portions of a protein) the stronger it will interact with the columnor the membrane. Thus, under suitable conditions, HIC can be used tofacilitate the separation of process-related impurities (e.g., HCPs) aswell as product-related substances (e.g., aggregates and fragments) froma protein of interest in a sample.

Like ion exchange chromatography, a HIC column or a HIC membrane devicecan also be operated in an elution mode, a flowthrough, or a hybrid modewherein the product exhibits binding to or interacting with achromatographic material yet can be washed from such material using abuffer that is the same or substantially similar to the loading buffer.(The details of these modes are outlined above in connection with AEXprocessing.) As hydrophobic interactions are strongest at high ionicstrength, this form of separation is conveniently performed following asalt elution step such as those typically used in connection with ionexchange chromatography. Alternatively, salts can be added to a samplebefore employing a HIC step. Adsorption of a protein to a HIC column isfavored by high salt concentrations, but the actual concentrations canvary over a wide range depending on the nature of the protein ofinterest, salt type and the particular HIC ligand chosen. Various ionscan be arranged in a so-called soluphobic series depending on whetherthey promote hydrophobic interactions (salting-out effects) or disruptthe structure of water (chaotropic effect) and lead to the weakening ofthe hydrophobic interaction. Cations are ranked in terms of increasingsalting out effect as Ba²⁺; Ca²⁺; Mg²⁺; Li⁺; Cs⁺; Na⁺; K⁺; Rb⁺; NH4⁺,while anions may be ranked in terms of increasing chaotropic effect asPO₄ ³⁻; SO₄ ²⁻; CH₃CO₃ ⁻; CI⁻; Br⁻; NO₃ ⁻; ClO₄ ⁻; I⁻; SCN⁻.

In general, Na⁺, K⁺ or NH4⁺ sulfates effectively promote ligand-proteininteraction using HIC. Salts may be formulated that influence thestrength of the interaction as given by the following relationship:(NH₄)₂SO₄>Na₂SO₄>NaCl>NH₄Cl>NaBr>NaSCN. In general, salt concentrationsof between about 0.75 M and about 2 M ammonium sulfate or between about1 M and about 4 M NaCl are useful.

HIC media normally comprise a base matrix (e.g., cross-linked agarose orsynthetic copolymer material) to which hydrophobic ligands (e.g., alkylor aryl groups) are coupled. A suitable HIC media comprises an agaroseresin or a membrane functionalized with phenyl groups (e.g., a PhenylSepharose™ from GE Healthcare or a Phenyl Membrane from Sartorius). ManyHIC resins are available commercially. Examples include, but are notlimited to, Capto Phenyl, Phenyl Sepharose™ 6 Fast Flow with low or highsubstitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ HighPerformance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMDPhenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butylcolumns (Bio-Rad, California); WP HI-Propyl (C3)™ (J. T. Baker, NewJersey); and Toyopearl™ ether, phenyl or butyl (TosoHaas, PA);ToyoScreen PPG; ToyoScreen Phenyl; ToyoScreen Butyl; ToyoScreen Hexyl;GE HiScreen and Butyl FF HiScreen Octyl FF.

The protein load of a sample comprising a protein of interest isadjusted to a total protein load to the column of between about 50 g/Lto about 1000 g/L; about 5 g/L and about 150 g/L, between about 10 g/Land about 100 g/L, between about 20 g/L and about 80 g/L, between about30 g/L and about 50 g/L, or between about 40 g/L and about 50 g/L. Incertain embodiments, the protein concentration of the load proteinmixture is adjusted to a protein concentration of the material to beloaded onto the column of about 0.5 g/L and about 50 g/L, or betweenabout 1 g/L and about 20 g/L.

Because the pH selected for any particular production process must becompatible with protein stability and activity, particular pH conditionsmay be specific for each application. However, because at pH 5.0-8.5particular pH values have very little significance on the finalselectivity and resolution of a HIC separation, such conditions may befavored. An increase in pH weakens hydrophobic interactions andretention of proteins changes more drastically at pH values above 8.5 orbelow 5.0. In addition, changes in ionic strength, the presence oforganic solvents, temperature and pH (especially at the isoelectricpoint, pI, when there is no net surface charge) can impact proteinstructure and solubility and, consequently, the interaction with otherhydrophobic surfaces, such as those in HIC media and hence, in certainembodiments, the present invention incorporates production strategieswherein one or more of the foregoing are adjusted to achieve the desiredreduction in process-related impurities and/or product-relatedsubstances.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, and Raman may be used to monitor the protein of interestand impurities in an on-line, at-line or in-line mode, which can then beused to control the level of aggregates in the pooled material collectedfrom the HIC adsorbent effluent. In certain embodiments, on-line,at-line or in-line monitoring methods can be used either on the effluentline of the chromatography step or in the collection vessel, to enableachievement of the desired product quality/recovery. In certainembodiments, the UV signal can be used as a surrogate to achieve anappropriate product quality/recovery, wherein the UV signal can beprocessed appropriately, including, but not limited to, such processingtechniques as integration, differentiation, and moving average, suchthat normal process variability can be addressed and the target productquality can be achieved. In certain embodiments, such measurements canbe combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback and hence facilitate product quality control.

G. Size Exclusion Chromatography

Size exclusion chromatography or gel filtration relies on the separationof components as a function of their molecular size. Separation dependson the amount of time that the substances spend in the porous stationaryphase as compared to time in the fluid. The probability that a moleculewill reside in a pore depends on the size of the molecule and the pore.In addition, the ability of a substance to permeate into pores isdetermined by the diffusion mobility of macromolecules which is higherfor small macromolecules. Very large macromolecules may not penetratethe pores of the stationary phase at all; and, for very smallmacromolecules the probability of penetration is close to unity. Whilecomponents of larger molecular size move more quickly past thestationary phase, components of small molecular size have a longer pathlength through the pores of the stationary phase and are thus retainedlonger in the stationary phase.

The chromatographic material can comprise a size exclusion materialwherein the size exclusion material is a resin or membrane. The matrixused for size exclusion is preferably an inert gel medium which can be acomposite of cross-linked polysaccharides, for example, cross-linkedagarose and/or dextran in the form of spherical beads. The degree ofcross-linking determines the size of pores that are present in theswollen gel beads. Molecules greater than a certain size do not enterthe gel beads and thus move through the chromatographic bed the fastest.Smaller molecules, such as detergent, protein, DNA and the like, whichenter the gel beads to varying extent depending on their size and shape,are retarded in their passage through the bed. Molecules are thusgenerally eluted in the order of decreasing molecular size.

Porous chromatographic resins appropriate for size-exclusionchromatography of viruses may he made of dextrose, agarose,polyacrylamide, or silica which have different physical characteristics.Polymer combinations can also be also used. Most commonly used are thoseunder the tradename, “SEPHADEX” available from Amersham Biosciences.Other size exclusion supports from different materials of constructionare also appropriate, for example Toyopearl 55F (polymethacrylate, fromTosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRadLaboratories, Hercules, Calif.).

The protein load of a sample comprising a protein of interest can beadjusted to a total protein load to the column of between about 50 g/Land about 1000 g/L; about 5 g/L and about 150 g/L, between about 10 g/Land about 100 g/L, between about 20 g/L and about 80 g/L, between about30 g/L and about 50 g/L, or between about 40 g/L and about 50 g/L. Incertain embodiments, the protein concentration of the load proteinmixture is adjusted to a protein concentration of the material to beloaded onto the column of between about 0.5 g/L and about 50 g/L, orbetween about 1 g/L and about 20 g/L.

H. Viral Filtration

Viral filtration is a dedicated viral reduction step in a productionprocess. This step is usually performed post chromatographic polishing.Viral reduction can be achieved via the use of suitable filtersincluding, but not limited to, Planova 20N™, 50 N or BioEx from AsahiKasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPV fromSartorius, or Ultipor DV20 or DV50™ filter from Pall Corporation. Itwill be apparent to one of ordinary skill in the art to select asuitable filter to obtain desired filtration performance.

I. Ultrafiltration/Diafiltration

Certain embodiments of the present invention employ ultrafiltration anddiafiltration to further concentrate and formulate a protein ofinterest. Ultrafiltration is described in detail in: Microfiltration andUltrafiltration: Principles and Applications, L. Zeman and A. Zydney(Marcel Dekker, Inc., New York, N.Y., 1996); and in: UltrafiltrationHandbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No.87762-456-9); the entire teachings of which are incorporated herein byreference. One filtration process is Tangential Flow Filtration asdescribed in the Millipore catalogue entitled “Pharmaceutical ProcessFiltration Catalogue” pp. 177-202 (Bedford, Mass., 1995/96), the entireteaching of which is incorporated herein by reference. Ultrafiltrationis generally considered to mean filtration using filters with a poresize of smaller than 0.1 μιη. By employing filters having such a smallpore size, the volume of sample can be reduced through permeation of thesample buffer through the filter membrane pores while proteins areretained above the membrane surface.

One of ordinary skill in the art can select an appropriate membranefilter device for the UF/DF operation. Examples of membrane cassettessuitable for the present invention include, but not limited to, Pellicon2 or Pellicon 3 cassettes with 10 kD, 30 kD or 50 kD membranes from EMDMillipore, Kvick 10 kD, 30 kD or 50 kD membrane cassettes from GEHealthcare, and Centramate or Centrasette 10 kD, 30 kD or 50 kDcassettes from Pall Corporation.

J. Exemplary Production Strategies

Primary recovery can proceed by sequentially employing pH reduction,centrifugation, and filtration to remove cells and cellular debris(including HCPs) from a production bioreactor harvest. The presentinvention is directed to subjecting a biological sample comprising aprotein of interest from the primary recovery to one or more productionsteps, including (in no particular order) AEX, CEX, SEC, HIC and/or MM.Certain aspects of the present invention include further processingsteps. Examples of additional processing procedures include ethanolprecipitation, isoelectric focusing, reverse phase HPLC, chromatographyon silica, chromatography on heparin Sepharose™, further anion exchangechromatography and/or further cation exchange chromatography,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation,hydroxyapatite chromatography, gel electrophoresis, dialysis, andaffinity chromatography (e.g., using Protein A or G, an antibody, aspecific substrate, ligand or antigen as the capture reagent). Incertain aspects, the column temperature (as well as other parameters)can be independently varied to improve the separation efficiency and/oryield of any particular production step.

In certain embodiments, unbound flowthrough and wash fractions can befurther fractionated and a combination of fractions providing a targetproduct purity can be pooled.

Column loading and washing steps can be controlled by in-line, at-lineor off-line measurement of the product related impurity/substancelevels, either in the column effluent, or the collected pool or both, soas to achieve a particular target product quality and/or yield. Incertain embodiments, the loading concentration can be dynamicallycontrolled by in-line or batch or continuous dilutions with buffers orother solutions to achieve the partitioning necessary to improve theseparation efficiency and/or yield.

Examples of such production procedures are depicted in FIGS. 5-8.

FIG. 5 represents one exemplary embodiment used for the production ofaflibercept. Referring to FIG. 5, the method comprises: (a) expressingaflibercept in a host cell cultured in a CDM; (b) capturing afliberceptusing a first chromatography support, which can include affinity captureresin; and (c) contacting at least a portion of aflibercept with asecond chromatography support, which can include anion-exchangechromatography. Step (c) can further comprise washing an AEX column andcollecting flowthrough fraction(s) of a sample comprising aflibercept.Optionally, step (c) can comprise stripping the second chromatographicsupport and collecting stripped fractions. The steps can be carried outby routine methodology in conjunction with methodology mentioned supra.It should be understood that one skilled in the art might opt to employCEX rather than or in addition to AEX. In no particular order,additional chromatographic steps may be employed as well including, butnot limited to, HIC and SEC.

In addition to the exemplary embodiment in FIG. 5, other additionalexemplary embodiments can include (d) contacting at least a portion ofsaid aflibercept of step (c) with a third chromatography support. In oneaspect, the protocol can include (e) contacting at least a portionaflibercept of step (d) with a fourth chromatography support. In oneaspect of this embodiment, the protocol can optionally comprisesubjecting the sample comprising aflibercept of step (c) to a pH lessthan 5.5. In one aspect, the present method comprises a clarificationstep prior to step (a).

FIG. 6 represents one exemplary embodiment used for the production ofVEGF MiniTrap. This method comprises: (a) expressing aflibercept in ahost cell cultured in a CDM; (b) capturing aflibercept using a firstchromatography support which can include affinity chromatography resin;(c) cleaving the aflibercept thereby removing the Fc domain and forminga sample comprising VEGF MiniTrap; (d) contacting the sample of step (c)with a second chromatographic support which can be affinitychromatography and (e) contacting the flowthrough of step (d) to a thirdchromatography support which can include an anion-exchangechromatography. Step (d) comprises the collection of flowthroughfraction(s) where due to the absence of an Fc domain, the MiniTrapshould reside while the aflibercept or any other protein having an Fcdomain should essentially interact with the affinity column of step (d).Optionally, step (d) can comprise stripping the third chromatographicsupport and collecting stripped fractions. The steps can be carried outby routine methodology in conjunction with methodology outlined above.In no particular order, additional chromatographic steps can be employedincluding, but not limited to, HIC and SEC.

FIG. 7 represents one exemplary embodiment for the production ofaflibercept. This method comprises: (a) expressing aflibercept in a hostcell cultured in a CDM; (b) capturing aflibercept using a firstchromatography support, which can include cation exchangechromatography; and (c) contacting a flowthrough of step (b) to a secondchromatography support which can include an anion-exchangechromatography. Optionally, step (c) can comprise stripping the secondchromatographic support and collecting stripped fractions. The steps canbe carried out by routine methodology in conjunction with protocolsalluded to above. In no particular order, other chromatographicprocedures may be employed including, but not limited to, HIC and SEC.

FIG. 8 represents one exemplary embodiment for producing VEGF MiniTrap.This method comprises: (a) expressing aflibercept in a host cellcultured in a CDM; (b) capturing aflibercept using a firstchromatography support which can include an ion exchange chromatography;(c) subjecting a flowthrough fraction of (b) comprising aflibercept toaffinity chromatography; eluting, wherein the elution comprisesaflibercept; (d) subjecting the aflibercept of (c) to a cleavageactivity, whereby the Fc domain is cleaved thus forming VEGF MiniTrap.In one aspect, the ion exchange of step (b) comprises AEX.Alternatively, step (b) may comprise CEX. In no particular order,additional chromatographic steps may be included such as further ionexchange chromatography steps following step (d), the addition of HICand/or SEC.

VII. Pharmaceutical Formulations Comprising the Compositions

The invention also discloses formulations comprising anti-VEGFcompositions (as described above). Suitable formulations for anti-VEGFproteins include, but are not limited to, formulations described in U.S.Pat. No. 7,608,261, U.S. Pat. No. 7,807,164, U.S. Pat. No. 8,092,803,U.S. Pat. No. 8,481,046, U.S. Pat. No. 8,802,107, U.S. Pat. No.9,340,594, U.S. Pat. No. 9,914,763, U.S. Pat. No. 9,580,489, U.S. Pat.No. 10,400,025, U.S. Pat. No. 8,110,546, U.S. Pat. No. 8,404,638, U.S.Pat. No. 8,710,004, U.S. Pat. No. 8,921,316, U.S. Pat. No. 9,416,167,U.S. Pat. No. 9,511,140, U.S. Pat. No. 9,636,400, and U.S. Pat. No.10,406,226, which are all incorporated herein by reference in theirentirety.

The upstream process technologies (described in Section IV, supra) anddownstream process technologies (described in Section V, supra) may beused alone or in combination with each other to effect formulationproduction.

The present invention discloses formulations comprising anti-VEGFcompositions in association with one or more ingredients/excipients aswell as methods of use thereof and methods of making such compositions.In an embodiment of the invention, a pharmaceutical formulation of thepresent invention has a pH of approximately 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1 or 6.2.

To prepare pharmaceutical formulations for anti-VEGF compositions, ananti-VEGF composition is admixed with a pharmaceutically acceptablecarrier or excipient. See, for example, Remington's PharmaceuticalSciences and U.S. Pharmacopeia: National Formulary, Mack PublishingCompany, Easton, Pa. (1984); Hardman, et al. (2001) Goodman and Gilman'sThe Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.;Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, MarcelDekker, Inc., New York, N.Y.; the entire teachings of which areincorporated herein by reference. In an embodiment of the invention, thepharmaceutical formulation is sterile.

Pharmaceutical formulations of the present invention include ananti-VEGF composition and a pharmaceutically acceptable carrierincluding, for example, water, buffering agents, preservatives and/ordetergents.

The present invention provides a pharmaceutical formulation comprisingany of the anti-VEGF compositions set forth herein and apharmaceutically acceptable carrier, for example, wherein theconcentration of polypeptide is about 40 mg/mL, about 60 mg/mL, about 80mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120mg/mL, about 130 mg/mL, about 140 mg/mL, about 150 mg/mL, about 200mg/mL or about 250 mg/mL.

The scope of the present invention includes desiccated, for example,freeze-dried, compositions comprising an anti-VEGF protein and apharmaceutically acceptable carrier substantially (about 85% to about99% or greater) lacking water.

In one embodiment, a further therapeutic agent that is administered to asubject in association with an anti-VEGF composition disclosed herein isadministered to the subject in accordance with the Physicians' DeskReference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002), theteaching of which is incorporated herein by reference).

The present invention provides a vessel (e.g., a plastic or glass vialwith a cap or a chromatography column, hollow bore needle or a syringecylinder) comprising any of the anti-VEGF compositions or apharmaceutical formulation comprising a pharmaceutically acceptablecarrier described herein. The present invention also provides aninjection device comprising the anti-VEGF composition or formulation setforth herein, for example, a syringe, a pre-filled syringe or anautoinjector. In one aspect, the vessel is tinted (e.g., brown) to blockout light, natural or otherwise.

The present invention includes combinations including anti-VEGFcompositions in association with one or more further therapeutic agents.The anti-VEGF composition and the further therapeutic agent can be in asingle composition or in separate compositions. For example, thetherapeutic agent is an Ang-2 inhibitor (e.g., nesvacumab), a Tie-2receptor activator, an anti-PDGF antibody or antigen-binding fragmentthereof, an anti-PDGF receptor or PDGF receptor beta antibody orantigen-binding fragment thereof and/or an additional VEGF antagonistsuch as aflibercept, conbercept, bevacizumab, ranibizumab, an anti-VEGFaptamer such as pegaptanib (e.g., pegaptanib sodium), a single chain(e.g., VL-VH) anti-VEGF antibody such as brolucizumab, an anti-VEGFDARPin such as the Abicipar Pegol DARPin, a bispecific anti-VEGFantibody, for example, which also binds to ANG2, such as RG7716, or asoluble form of human vascular endothelial growth factor receptor-3(VEGFR-3) comprising extracellular domains 1-3, expressed as anFc-fusion protein.

VIII. Methods of Treatment

The present invention provides methods for treating or preventing acancer (e.g., whose growth and/or metastasis is mediated, at least inpart, by VEGF, for example, VEGF-mediated angiogenesis) or an angiogeniceye disorder, in a subject, comprising administering a therapeuticallyeffective amount of compositions as disclosed herein (Section IIIsupra).

Upstream process technologies (Section IV supra), downstream processtechnologies (Sections V and VI supra) may be used alone or incombination with the each other to produce the compositions as describedin Section III and/or the formulations as described in Section VII whichcan be used for treating or preventing a variety of disorders includingophthalmological and oncological disease.

The present invention also provides a method for administeringcompositions set forth herein (Section III and Section VII) to a subject(e.g., a human) comprising introducing the compositions with about 0.5mg, 2 mg, 4 mg, 6 mg, 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg or 20 mgof the protein of interest (e.g., aflibercept or MiniTrap) in no morethan about 100 μL, for example, about 50 μL, about 70 μL or about 100μL, and optionally a further therapeutic agent, into the body of thesubject by, for example, intraocular injection such as by intravitrealinjection.

The present invention provides a method for treating cancer whose growthand/or metastasis is mediated, at least in part, by VEGF, for example,VEGF-mediated angiogenesis or an angiogenic eye disorder in a subject inneed thereof, the method comprising administering a therapeuticallyeffective amount of the compositions set forth herein (Section III andSection VII above), for example, 2 mg, 4 mg, 6 mg, 8 mg or 10 mg of theprotein of interest, in no more than about 100 μl, and optionally afurther therapeutic agent, to a subject. In one embodiment of theinvention, administration is done by intravitreal injection.Non-limiting examples of angiogenic eye disorders that are treatable orpreventable using the methods herein, include:

-   -   age-related macular degeneration (e.g., wet or dry),    -   macular edema,    -   macular edema following retinal vein occlusion,    -   retinal vein occlusion (RVO),    -   central retinal vein occlusion (CRVO),    -   branch retinal vein occlusion (BRVO),    -   diabetic macular edema (DME),    -   choroidal neovascularization (CNV),    -   iris neovascularization,    -   neovascular glaucoma,    -   post-surgical fibrosis in glaucoma,    -   proliferative vitreoretinopathy (PVR),    -   optic disc neovascularization,    -   corneal neovascularization,    -   retinal neovascularization,    -   itreal neovascularization,    -   pannus,    -   pterygium,    -   vascular retinopathy,    -   diabetic retinopathy in a subject with diabetic macular edema;        and    -   diabetic retinopathies (e.g., non-proliferative diabetic        retinopathy (e.g., characterized by a Diabetic Retinopathy        Severity Scale (DRSS) level of about 47 or 53) or proliferative        diabetic retinopathy; e.g., in a subject that does not suffer        from DME).

The mode of administration of such compositions or formulations (SectionIII and Section VII) can vary and can be determined by a skilledpractitioner. Routes of administration include parenteral,non-parenteral, oral, rectal, transmucosal, intestinal, parenteral,intramuscular, subcutaneous, intradermal, intramedullary, intrathecal,direct intraventricular, intravenous, intraperitoneal, intranasal,intraocular, inhalation, insufflation, topical, cutaneous, intraocular,intravitreal, transdermal or intra-arterial.

In one embodiment of the invention, intravitreal injection of apharmaceutical formulation of the present invention (which includes acompositions or formulations of the present invention) includes the stepof piercing the eye with a syringe and needle (e.g., 30-gauge injectionneedle) comprising the formulation and injecting the formulation (e.g.,less than or equal to about 100 microliters; about 40, 50, 55, 56, 57,57.1, 58, 60 or 70 microliters) into the vitreous of the eye with asufficient volume as to deliver a therapeutically effective amount asset forth herein, for example, of about 2, 4, 6, 8.0, 8.1, 8.2, 8.3,8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 10 or 20 mg of the protein of interest.Optionally, the method includes the steps of administering a localanesthetic (e.g., proparacaine, lidocaine or tetracaine), an antibiotic(e.g., a fluoroquinolone), antiseptic (e.g., povidone-iodine) and/or apupil dilating agent to the eye being injected. In one aspect, a sterilefield around the eye to be injected is established before the injection.Following intravitreal injection, the subject is monitored forelevations in intraocular pressure, inflammation and/or blood pressure.

An effective or therapeutically effective amount of protein of interestfor an angiogenic eye disorder refers to the amount of the protein ofinterest sufficient to cause the regression, stabilization orelimination of the cancer or angiogenic eye disorder, for example, byregressing, stabilizing or eliminating one or more symptoms or indiciaof the cancer or angiogenic eye disorder by any clinically measurabledegree, for example, with regard to an angiogenic eye disorder, bycausing a reduction in or maintenance of diabetic retinopathy severityscore (DRSS), by improving or maintaining vision (e.g., in bestcorrected visual acuity as measured by an increase in ETDRS letters),increasing or maintaining visual field and/or reducing or maintainingcentral retinal thickness and, with respect to cancer, stopping orreversing the growth, survival and/or metastasis of cancer cells in thesubject.

In one embodiment of the invention, an effective or therapeuticallyeffective amount of a protein of interest such as aflibercept fortreating or preventing an angiogenic eye disorder is about 0.5 mg, 2 mg,3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 7.25 mg, 7.7 mg, 7.9 mg, 8.0 mg, 8.1 mg,8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 10mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20mg, e.g., in no more than about 100 μL. The amount may vary dependingupon the age and the size of a subject to be administered, targetdisease, conditions, route of administration, and the like. In certainexemplary embodiments, the initial dose may be followed byadministration of a second or a plurality of subsequent doses of theprotein of interest in an amount that can be approximately the same orless or more than that of the initial dose, wherein the subsequent dosesare separated by at least 1 day to 3 days; at least one week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10weeks, at least 12 weeks, or at least 14 weeks.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

IX. Method of Assaying Protein Variants

The levels of protein variants in a chromatographic sample producedusing the techniques described herein may be analyzed as described inthe Examples below. In certain embodiments, a cIEF method is employedusing an iCE3 analyzer (ProteinSimple) with a fluorocarbon coatedcapillary cartridge (100 μπι×5 cm). The ampholyte solution consists of amixture of 0.35% methyl cellulose (MC), 4% Pharmalyte 3-10 carrierampholytes, 4% Pharmalyte 5-8 carrier ampholytes, 10 mM L-Arginine HCl,24% formamide, and pI markers 5.12 and 9.77 in purified water. Theanolyte was 80 mM phosphoric acid, and the catholyte was 100 mM sodiumhydroxide, both in 0.10% methylcellulose. Samples were diluted inpurified water to 10 mg/mL. Samples were mixed with the ampholytesolution and then focused by introducing a potential of 1500 V for oneminute, followed by a potential of 3000 V for 7 minutes. An image of thefocused variants was obtained by passing 280 nm ultraviolet lightthrough the capillary and into the lens of a charge coupled devicedigital camera. This image was then analyzed to determine thedistribution of the various charge variants. Persons of skill in the artmay vary the precise parameters while still achieving the desiredoutcome.

Various publications, including patents, patent applications, publishedpatent applications, accession numbers, technical articles and scholarlyarticles are cited throughout the specification. Each of these citedreferences is incorporated by reference, in its entirety.

The present invention will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingthe scope of the invention.

EXAMPLES

The MiniTraps (MT) 1-6 discussed in the Examples are as follows:

-   MT1: VEGF MiniTrap obtained by cleavage of aflibercept produced    using CDM1.-   MT2: VEGF MiniTrap obtained by cleavage of aflibercept produced    using CDM2.-   MT3: VEGF MiniTrap obtained by cleavage of aflibercept produced    using CDM3.-   MT4: VEGF MiniTrap obtained by cleavage of aflibercept produced    using soy hydrolysate.-   MT5: recombinant VEGF MiniTrap (dimer).-   MT6: recombinant VEGF MiniTrap (scFv).    Characterization of MT1, MT5 and MT6 are described below in Example    8.

Color Assessment of Samples

The spectrophotometric assay method of measuring the b* value (CIELAB)was found suitable for performing color assessment.

The absorbance of a 1 mL protein sample was quantified over the visiblelight spectrum (380 to 780 nm) and the absorbance curve was transformedinto the CIELAB color space using a set of matrix operations. Theinstrument can process approximately 6 samples per hour. The highthroughput format of the assay used a CLARIOstar plate reader (BMGLabtech). Up to 96 samples can be analyzed using a 96-well platerequiring 0.3 mL of sample.

To convert the BY standards into the b* values, BY reference standards(BY1 to BY7) were quantified using the high throughput assay format.

The solutions were prepared as per the BY standards discussed above. Theb* value for each of the standards are as shown in FIG. 9. This methodprovided a faster assay with a smaller sample requirement and shorterrun times as shown in Table 3 below. For all the samples evaluated usingthis method, the protein concentration of the test samples wasstandardized to either 5 g/L or 10 g/L.

TABLE 3 Original High-throughput Amount/Sample 1 mL 0.3 mL MeasurementFormat cuvette (individual) 96-well-plate (bulk) Run Time 6 samples perhour 96 samples per 5 minutes Data Entry manual automated Data StorageExcel LIMS

Example 1 Production of a Protein Using a Chemically Defined Medium 1.1Cell Source and Harvest

An aflibercept producing cell line was employed in the present study.Aflibercept producing cell lines were cultured and harvested usingchemically defined media (CDM).

1.2 Proteolytic Cleavage of Aflibercept

A column with an immobilized IdeS enzyme (FabRICATOR® obtained fromGenovis (Cambridge, Mass.)) was used to generate MT1. Afliberceptobtained from a cell culture harvest (20 mg in 1.0 mL cleavage buffer)was added to the column and incubated for 30 min at 18° C. After 30 min,the column was washed with the cleavage buffer (1.0 mL). The digestionmixture and washing solutions were combined. The mixture was loaded ontoand eluted from an analytical Protein A affinity column (AppliedBiosystems™, POROS™ 20 μM Protein A Cartridge 2.1×30 mm, 0.1 mL (Cat#2-1001-00)). The processing was carried out according to AppliedBiosystems'™ protocol for POROS™ 20 μM Protein A Cartridge 2.1×30 mm,0.1 mL (Cat #2-1001-00). The column height was 20±1.0 cm, residence timewas 15 minutes and equilibration/wash was performed using 40 mM Tris, 54mM Acetate pH 7.0±0.1.

Example 2 Anion Exchange Chromatography (AEX) for Color Minimization (A)AEX was Employed to Reduce Color Formation

AEX chromatography was performed to remove the coloration obtainedduring production of aflibercept expressed using CDM1.

2.1 Design

Five AEX separations were performed for this study as detailed in Table2-1 with the AEX protocol as described in Table 2-2. A 15.7 mL QSepharose Fast Flow column (19.5 cm bed height, 1.0 cm I.D.) and a 14.1mL POROS 50 HQ column (18.0 cm bed height, 1.0 cm I.D.) were integratedinto an AKTA Avant benchtop liquid chromatography controller.

AEX load pH was adjusted to target±0.05 pH units using 2 M Tris base or2 M acetic acid. AEX load conductivity was adjusted to target±0.1 mS/cmusing 5 M sodium chloride or deionized water. All pool samples wereanalyzed for high molecular weight (HMW), color and yield.

TABLE 2-1 Summary of the Study Design for AEX Color Reduction AEXSeparation Condition Evaluated Resin 1 pH 8.30-8.50, 1.90-2.10 mS/cmPOROS 50 HQ 2 pH 7.90-8.10, 2.40-2.60 mS/cm Q Sepharose FF 3 pH7.90-8.10, 2.40-2.60 mS/cm POROS 50 HQ 4 pH 7.70-7.90, 3.90-4.10 mS/cm QSepharose FF 5 pH 7.70-7.90, 3.90-4.10 mS/cm POROS 50 HQ

TABLE 2-2 AEX Protocol for Color Reduction Column Linear VolumesVelocity Step Description Mobile Phase (CVs) (cm/h) 1 Pre- 2M SodiumChloride (NaCl) 2 200 Equilibration 2 Equilibration 50 mM Tris, VariablemM NaCl 2 200 Variable pH and Conductivity 3 Load AEX Load Variable 40g/L- 200 pH and Conductivity resin 4 FT/Wash 50 mM Tris, Variable mMNaCl 2 200 Variable pH and Conductivity 5 Strip 1 2M Sodium Chloride(NaCl) 2 200 6 Strip 2 1N Sodium Hydroxide (NaOH) 2 200

2.2 Results

Employing AEX separations for production exhibited a significantreduction in color. (Table 2-3). For example, as seen in Table 2-3, thecolor observed in the flowthrough (FT) and wash in AEX separation 1 (pH8.30-8.50, 1.90-2.10 mS/cm) had a b* value of 1.05, as compared to thecolor of the Load for AEX (“AEX Load”) with a b* value of 3.06. Theincrease in b* value reflects the intensity of yellow-brown colorationof a sample.

Five AEX separations were performed to evaluate the impact of resin (QSepharose FF or POROS 50 HQ) and pH and conductivity setpoint (pH 8.40and 2.00 mS/cm, pH 8.00 and 2.50 mS/cm, or pH 7.80 and 4.00 mS/cm) oncolor reduction. For POROS 50 HQ, yields (64.4, 81.9, and 91.4%) andpool HMW levels (1.02, 1.29, and 1.83%) increased as the setpoint waschanged to a lower pH and higher conductivity. Color (b* values) alsoincreased (1.05, 1.33, and 1.55) as the setpoint was changed to a lowerpH and higher conductivity. The higher pH levels and lowerconductivities provided the most reduction in color over the AEXseparation for POROS 50 HQ.

For Q Sepharose Fast Flow, yields (49.5 and 77.7%) and pool HMW levels(0.59 and 1.25%) also increased as the setpoint was changed to a lowerpH and higher conductivity. Color (b* values) also increased (0.96 and1.35) as the setpoint was changed to a lower pH and higher conductivity.

The use of AEX reduces yellow-brown coloration—see Table 2-3.Additionally, it was determined that Q Sepharose Fast Flow reduced colormore than POROS 50 HQ for the two set points evaluated on both resins.At pH 8.00 and 2.50 mS/cm setpoint, POROS 50 HQ pool had a b* value of1.33 while Q Sepharose Fast Flow pool had a b* value of 0.96. Similarly,at pH 7.80 and 4.00 mS/cm setpoint, POROS 50 HQ pool had a b* value of1.55 while Q Sepharose Fast Flow pool had a b* value of 1.35 (Table2-3).

TABLE 2-3 Summary of Experimental Results of the AEX Color ReductionStudy AEX Yield HMW Color Color Color Separation Fraction (%) (%) (L*)(a*) (b*) 1 FT/wash 64.4 1.02 98.89  0.01 1.05 2 FT/wash 49.5 0.59 98.30−0.03 0.96 3 FT/wash 81.9 1.29 99.07 −0.07 1.33 4 FT/wash 77.7 1.2599.42 −0.04 1.35 5 FT/wash 91.4 1.83 99.19 −0.09 1.55 — filtered pool —3.66 98.73 −0.21 3.06 (AEX Load) 3.98 AEX, anion exchangechromatography; HMW, high molecular weight species; N/A, not applicableThe fractions were adjusted to a protein concentration of 10 g/L forcolor measurements.

2.3 Conclusion

Use of AEX was found to reduce the yellow-brown coloration, see Table2-3. Referring to Table 2-3, the AEX Load has a b* value of 3.06, butwhen subjected to AEX chromatography (AEX Separation 1-5), the b* valuedecreases indicating a decrease in yellow-brown coloration. Again, asthe b* value decreases so does the coloration; as the b* value increasesit is reflective of the yellow-brown color increasing in a given sample.

Color reduction was evaluated using two AEX resins (POROS 50 HQ and QSepharose Fast Flow) and three set points (pH 8.40 and 2.00 mS/cm, pH8.00 and 2.50 mS/cm, and pH 7.80 and 4.00 mS/cm). For both resins, colorreduction was higher for the higher pH and lower conductivity setpoints. In addition, Q Sepharose Fast Flow provided more color reductionthan POROS 50 HQ at the two set points evaluated on both resins (pH 8.00and 2.50 mS/cm and pH 7.80 and 4.00 mS/cm). However, all the five AEXseparation methods led to a significant color reduction when compared tothe loading solution for AEX (“AEX Load”), demonstrating the importanceof AEX production in the process of aflibercept production expressedusing a CDM. The initial b* value of the AEX Load (at a concentration of10 g/L) may range from about 0.5 to about 30, more particularly fromabout 1.0 to about 25.0, and even more particularly from about 2.0 toabout 20.0. Following use of AEX, the b* value for the flowthrough (at aconcentration of 10 g/L) may range from 0.5 to about 10.0, moreparticularly from about 0.5 to about 7.0, and even more particularlyfrom about 0.5 to about 5.0.

2.4 Peptide Mapping

Sample preparation. Tryptic mapping of reduced and alkylated afliberceptsamples obtained from AEX Load and flowthrough of the above experiment(Table 2-3) were performed to identify and quantify post-translationalmodification (PTM). An aliquot of each sample (Load and flowthrough) wasdenatured using 8.0 M Urea, 0.1 M Tris-HCl, pH 7.5, reduced with DTT andthen alkylated with iodoacetamide. The denatured, reduced and alkylatedsample was first digested with recombinant Lys-C (rLys-C) at an enzymeto substrate ratio of 1:100 (w/w) at 37° C. for 30 minutes, diluted with0.1 M Tris-HCl, pH 7.5 such that a final urea concentration was 1.8 M,subsequently digested with trypsin at an enzyme to substance ratio of1:20 (w/w) at 37° C. for 2 hours and then deglycosylated with PNGase Fat an enzyme substrate ratio of 1:5 (w/w) for 37° C. for 1 hour. Thedigestion was stopped by bringing the pH below 2.0 using formic acid(FA).

LC-MS analysis. A 20 μg aliquot of resulting rLys-C/tryptic peptidesfrom each sample was separated and analyzed by reverse-phaseultra-performance liquid chromatography (UPLC) using Waters ACQUITY UPLCCSH C18 column (130 Å, 1.7 μm, 2.1×150 mm) followed by on-line PDAdetection (at wavelengths of 280 nm, 320 nm and 350 nm) and massspectrometry analysis. Mobile phase A was 0.1% FA in water, and mobilephase B was 0.1% FA in acetonitrile. After sample injection, a gradientwas initiated with a 5 minute hold at 0.1% B followed by a linearincrease to 35% B over 75 minutes for optimum peptide separation. MS andMS/MS experiments were conducted using a Thermo Scientific Q ExactiveHybrid Quadrupole-Orbitrap mass spectrometer with higher-energycollisional dissociation (HCD) employed for peptide fragmentation forMS/MS experiments. Peptide identity assignments were based on theexperimentally determined accurate mass of a given peptide in the fullMS spectrum as well as the b and y fragment ions in the correspondingHCD MS/MS spectrum. Extracted ion chromatograms of the peptides from theLoad and flowthrough were generated (see FIG. 10). As seen in theextracted ion chromatogram in FIG. 10, the peptide fragments identifiedin “AEX Load” and “AEX FT/wash” from AEX separations 1-5 (as identifiedin Table 2-3) are shown. The relative abundance of some of thesepeptides identified in FIG. 10 from the peptide mapping analysis areshown in FIG. 11.

Referring to FIG. 11, this figure identifies various peptide fragmentsanalyzed and their relative levels of oxidation. In particular, thethird column identifies the amino acid residues (“Peptide Sequence”) ofpeptide fragments that were isolated and analyzed. Each Peptide Sequencehas an amino acid residue that is underscored. The underscored aminoacid residue identifies the amino acid in the Peptide Sequence that isoxidized. The oxidized amino acids correspond to either histidine (H)oxidation or tryptophan (W) oxidation. There is also depicted in thisfigure rows to the right of each of the Peptide Sequences showing theabundance of oxidized species. This shading in the rows indicatesdifferences in the relative amount of oxidized residues in a particularsample using different AEX separations identified in the respectivecolumn headings. For example, referring to the second peptide,EIGLLTCEATVNGHLYK (SEQ ID NO.: 18) in FIG. 11, when read across along ina horizontal manner, the relative total population of this peptide in aparticular sample (“aflibercept AEX Load”) that is oxidized isapproximately 0.013% oxidized. As progression is made across the samerow, there is a shift in the shading, indicating a change in therelative abundance of oxidized species. For example, using this samePeptide Sequence, the relative abundance of oxidized species for AEXseparation are 0.006% to 0.010% when following different AEX separationprotocols. Thus, it can be appreciated that AEX chromatography decreasesthe abundance of oxidized species.

(B) AEX was Employed to Reduce the Color Formation in MiniTrapProduction

AEX chromatography was performed to remove the coloration obtainedduring production of MT1 which was obtained on performing cleavage offull-length aflibercept expressed using CDM1.

2.5 Design

Four AEX separations were performed for this study as described in Table2-4. The AEX Load was obtained from a filtration sample of MT1 (“MT1filtered pool”). A 15.7 mL Capto Q column (20.0 cm bed height, 1.0 cmI.D.), a 14.1 mL POROS 50 HQ column (18.0 cm bed height, 1.0 cm I.D.),and a 16.5 mL Q Sepharose FF column (21.0 cm bed height, 1.0 cm I.D.)were integrated into an AKTA Avant benchtop liquid chromatographycontroller for this experiment.

AEX load pH was adjusted to target±0.05 pH units using 2 M tris base or2 M acetic acid. AEX load conductivity was adjusted to target±0.1 mS/cmusing 5 M sodium chloride or deionized water. All pool samples wereanalyzed for HMW, color and yield.

TABLE 2-4 Summary of the Study Design for the AEX Color Reduction StudyAEX Separation Resin AEX Protocol 1 Capto Q Table 2-6 2 POROS 50 HQTable 2-6 3 Q Sepharose FF Table 2-6 4 POROS 50 HQ Table 2-5

TABLE 2-5 Flowthrough AEX Protocol Used for the Color Reduction StudyColumn Linear Volumes Velocity Step Description Mobile Phase (CVs)(cm/h) 1 Pre- 2M Sodium Chloride (NaCl) 2 200 Equilibration 2Equilibration 50 mM Tris, 40 mM NaCl 2 200 pH 7.90-8.10, 6.50-7.50 mS/cm3 Load AEX Load 30 g/L- 200 pH 7.90-8.10, 6.50-7.50 mS/cm resin 4 Wash50 mM Tris, 40 mM NaCl 2 200 pH 7.90-8.10, 6.50-7.50 mS/cm 5 Strip 1 2MSodium Chloride (NaCl) 2 200 6 Strip 2 1N Sodium Hydroxide (NaOH) 2 200AEX, anion exchange chromatography; CV, column volume

TABLE 2-6 Bind and Elute AEX Protocol Used for the Color Reduction StudyColumn Linear Volumes Velocity Step Description Mobile Phase (CVs)(cm/h) 1 Pre- 2M Sodium Chloride (NaCl) 2 200 Equilibration 2Equilibration 50 mM Tris 2 200 pH 8.30-8.50, 1.90-2.10 mS/cm 3 Load AEXLoad 30 g/L- 200 pH 8.30-8.50, 1.90-2.10 mS/cm resin 4 Wash 50 mM Tris 2200 pH 8.30-8.50, 1.90-2.10 mS/cm 5 Elution 50 mM Tris, 70 mM NaCl 2 200pH 8.30-8.50, 8.50-9.50 mS/cm 6 Strip 1 2M Sodium Chloride (NaCl) 2 2007 Strip 2 1N Sodium Hydroxide (NaOH) 2 200 AEX, anion exchangechromatography; CV, column volume

2.6 Results

All four AEX separations led to reduction in color as seen forcoloration of the flowthrough and wash for AEX separations 1-4 (Table2-7). While the first three AEX separations were evaluated in a bind andelute mode (Table 2-6), it was observed that the majority of the productwas present in the load and wash blocks (62%-94%).

The first three separations evaluated the pH 8.4 and 2.0 mS/cm setpointfor Capto Q, POROS 50 HQ, and Q Sepharose FF resins. All threeseparations had a good yield (>80%). The POROS 50 HQ AEX pool showed thelowest yellow color in AEX pool (b* value of 2.09) followed by the QSepharose FF AEX pool (b* value of 2.22) and the Capto Q AEX pool (b*value of 2.55).

TABLE 2-7 Summary of Experimental Results of the AEX Color ReductionStudy AEX Yield HMW Color Color Color Separation Fraction (%) (%) (L*)(a*) (b*) 1 FT/wash 90.7 0.49 99.11 −0.27 2.55 2 FT/wash 93.8 0.33 99.20−0.28 2.09 3 FT/wash 86.7 0.23 98.88 −0.23 2.22 4 FT/wash 99.5 1.1398.90 −0.39 3.40 — MT1 Filtered Pool — 0.65 98.18 −0.37 4.17 (AEX Load)AEX, anion exchange chromatography; HMW, high molecular weight species.The fractions were adjusted to a protein concentration of 5 g/L forcolor measurements.

2.7 Conclusion

As seen for aflibercept (see Section 2.3 above), use of AEX was found toreduce yellow-brown coloration (Table 2-7) for MiniTrap production.Referring to Table 2-7, the AEX Load has a b* value of 4.17, but whensubjected to AEX chromatography (AEX Separation 1-4), the b* valuedecreases indicating a decrease in yellow-brown coloration. Again, asthe b* value decreases so too does the coloration. The initial b* valueof the AEX Load (at a concentration of 5 g/L) may range from about 0.5to about 25, more particularly from about 1.0 to about 20.0, and evenmore particularly from about 1.5 to about 15.0. Following use of AEX,the b* value of the flowthrough (at a concentration of 5 g/L) may rangefrom 0.5 to about 10.0, more particularly from about 0.5 to about 7.0,and even more particularly from about 0.5 to about 5.0.

Example 3 Oxidized Peptide Study

3.1 Peptide Mappings

Sample Preparation. Tryptic mapping of reduced and alkylated MiniTrap(MT1) and MT4 (MiniTrap similar to MT1 using a different full-lengthaflibercept one produced using soy hydrolysate cell culture) sampleswere performed to identify and quantify post-translational modification.An aliquot of sample was denatured using 8.0 M Urea in 0.1 M Tris-HCl,pH 7.5, reduced with DTT and then alkylated with iodoacetamide. Thedenatured, reduced and alkylated drug substance was first digested withrecombinant Lys-C (rLys-C) at an enzyme to substrate ratio of 1:100(w/w) at 37° C. for 30 minutes, diluted with 0.1 M Tris-HCl, pH 7.5 suchthat the final urea concentration was 1.8 M, subsequently digested withtrypsin at an enzyme to substance ratio of 1:20 (w/w) at 37° C. for 2hours and then deglycosylated with PNGase F at an enzyme substrate ratioof 1:5 (w/w) for 37° C. for 1 hour. The digestion was stopped bybringing the pH below 2.0 using formic acid (FA).

LC-MS Analysis. A 20 μg aliquot of resulting rLys-C/tryptic peptidesfrom each sample was separated and analyzed by reverse-phaseultra-performance liquid chromatography (UPLC) using Waters ACQUITY UPLCCSH C18 column (130 Å, 1.7 μm, 2.1×150 mm) followed by on-line PDAdetection (at wavelengths of 280 nm, 320 nm and 350 nm) and massspectrometry analysis. Mobile phase A was 0.1% FA in water and mobilephase B was 0.1% FA in acetonitrile. After sample injection, a gradientstarted with a 5 min hold at 0.1% B followed by a linear increase to 35%B over 75 minutes for optimum peptide separation. MS and MS/MSexperiments were conducted on a Thermo Scientific Q Exactive HybridQuadrupole-Orbitrap mass spectrometer with higher-energy collisionaldissociation (HCD) employed for peptide fragmentation for MS/MSexperiments. Peptide identity assignments were based on theexperimentally determined accurate mass of a given peptide in the fullMS spectrum as well as the b and y fragment ions in the correspondingHCD MS/MS spectrum. Extracted ion chromatograms of oxidized peptides andcorresponding native peptide were generated with the peak areasintegrated to calculate the site-specific percentage of oxidized aminoacid residue(s) within the MT1 sample.

Peptide Fragments Linked to Increased Absorbance at 350 nm

The PTMs on MT1 were observed upon comparing the tryptic peptide mapsfor MT1 and MT4 (FIG. 12A shows the absorbance of peptides eluted from20.0 to 75 minutes). The peptides with varying UV peaks are highlighted.The expanded view of the chromatogram is shown in FIG. 12B which showsthe absorbance of peptides eluted from 16 to 30 minutes. The peptideswith sharp contrast in UV absorbance between MT1 and MT4 were TNYLTH*R(SEQ ID NO.: 21), IIW(+4)DSR (SEQ ID NO.: 28) and IIIW(+132)DSR (SEQ IDNO.: 124) (* or underscoring represents oxidation of the residue).Further, the expanded view of the chromatogram is shown in FIG. 12C,which shows the absorbance of peptides eluted from 30 to 75 minutes. Thepeptides with sharp contrast in UV absorbance between MT1 and MT4 wereDKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), TELNVGIDFNWEYPSSKH*QHK (SEQ IDNO.: 20), EIGLLTCEATVNGH*LYK (SEQ ID NO.: 18) andQTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) (* represents oxidation of theresidue). The peptide mapping revealed identity of peptides that aresignificantly different in abundance between the VEGF MiniTraps. Therelative abundance of the peptides identified from the peptide mappinganalysis is shown in Table 3-1. The amount of 2-oxo-histidines in MT1(produced in a CDM) were higher than MT4 (produced in soy hydrolysate),suggesting that the media used to express aflibercept can have asignificant effect on the relative abundance of peptides with oxidizedhistidines or oxidized tryptophans. For example, for the peptideQTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), the percent relativeabundance of the peptide in MT1 (CDM produced) was 0.015% compared topercent relative abundance of the peptide in MT4 (soy hydrolysateproduced; which is about 15-fold less as compared to MT1).

TABLE 3-1 Peptide Modified Fold change Peptide Sequence MT1 MT4 MT1/MT4EIGLLTCEATVNGH EIGLLTC[+57]EATVN 0.011% 0.004% 2.75 LYK (SEQ IDGH[+14]LYK (SEQ ID NO.: 57) NO.: 18) QTNTIIDVVLSPSH QTNTIIDVVLSPSH[+14]0.015% 0.001% 15.00 GIELSVGEK (SEQ GIELSVGEK (SEQ ID NO.: 58)ID NO.: 19) TELNVGIDFNWEYP TELNVGIDFNWEYPS 0.204% 0.026% 7.85SSKHQHK (SEQ ID SKH[+14]QHK (SEQ ID NO.: 59) NO.: 20) DKTHTCPPCPAPELDKTH[+14]TC[+57]PP 0.115% 0.018% 6.39 LG (SEQ ID NO.: 60)C[+57]PAPELLG (SEQ ID NO.: 17) TNYLTHR (SEQ ID TNYLTH[+14]R (SEQ 0.130%0.020% 6.50 NO.: 61) ID NO.: 21)

Color and 2-oxo-Histidine Quantitation. The percentage of2-oxo-histidines in the oligopeptides that were generated by proteasedigestion, as measured by mass spectrometry, are also shown (Table 3-2).(Values were normalized against unmodified peptides.) Table 3-2 (I)shows the percent of oxidized histidines/tryptophans observed for AEXflowthrough: MT1 lot 1, AEX flowthrough for MT1 lot 2, and AEXflowthrough for MT1 lot 3. Table 3-2 (II) shows the percent of oxidizedhistidines/tryptophans observed for acidic fraction 1, acidic fraction2, and main fraction obtained on performing CEX separation for MT1 lot3. From this Table, it is clear that the acidic variants are comprisedof oxidized species. From Table 3-2(I), it is clear that the % of2-oxo-histidines and tryptophan dioxidation comprising peptides/proteinis reduced in the AEX flowthrough compared to the AEX Strip. It isevident that stripping the AEX column enriches for the percentage ofsuch modified peptides. For example, the % of the modified peptide“EIGLLTC[+57]EATVNGH[+14]LYK (SEQ ID NO.: 18)” in the AEX Flowthrough(MT1 lot 1) was 0.013% and in the “AEX Strip” was 0.080%. This alsocorroborates that the AEX column captures modified peptides, thusreducing the percentage of modified peptides in the AEX flowthrough.

TABLE 3-2 (I) Percentage of 2-oxo-Histidines/Tryptophans AEX FlowthroughAEX Strip MT1 lot1 MT1 lot 2 MT1 lot 3 Intense BY1, 110 ≤BY3, 110≤BY3, 110 Modified Peptides yellow mg/mL mg/mL mg/mLEIGLLTC[+57]EATVNGH[+14]LYK 0.080% 0.013% 0.008% 0.006% (SEQ ID NO.: 18)QTNTIIDVVLSPSH[+14]GIELSVGEK 0.054% 0.028% 0.023% 0.019%(SEQ ID NO.: 19) TELNVGIDFNWEYPSSKH[+14]QHK 0.235% 0.085% 0.049% 0.049%(SEQ ID NO.: 20) DKTH[+14]TC[+57]PPC[+57]PAPELLG 0.544% 0.092% 0.077%0.057% (SEQ ID NO.: 17) TNYLTH[+14]R (SEQ ID NO.: 21) 0.089% 0.022%0.011% 0.010% IIW[32]DSR (SEQ ID NO.: 28) 0.738% 0.252% 0.198% 0.298%

TABLE 3-2 (II) Percentage of 2-oxo-Histidines/TryptophansCEX flowthrough Acidic fraction 1 Acidic fraction 2 Main fractionfrom MT1 lot 3 from MT1 lot 3 from MT1 lot 3 Modified Peptides YellowYellow No Color EIGLLTC[+57]EATVNGH[+14]LYK 0.009% 0.008% 0.004%(SEQ ID NO.: 18) QTNTIIDVVLSPSH[+14]GIELSVGEK 0.013% 0.015% 0.006%(SEQ ID NO.: 19) TELNVGIDFNWEYPSSKH[+14]QHK 0.131% 0.151% 0.049%(SEQ ID NO.: 20) DKTH[+14]TC[+57]PPC[+57]PAPELLG 0.117% 0.132% 0.068%(SEQ ID NO.: 17) TNYLTH[+14]R (SEQ ID NO.: 21) 0.014% 0.008% 0.008%IIW[+32]DSR (SEQ ID NO.: 28) 0.458% 0.269% 0.185%

In Table 3-2(11), [+57] represent alkylation of cysteine byiodoacetamide, which adds a carboxymethyl amine moiety on the cysteine,which is a net mass increase of about +57 over unmodified cysteine:

In Table 3-2(11), [+14] represent conversion from His to 2-oxo-His. Oneoxygen atom is added on carbon 2, but two hydrogen atoms are lost (onefrom Carbon 2, the other from nitrogen 3), which is a net mass increaseof about +14 over unmodified histidine.

In Table 3-2(11), [+32] represents tryptophan dioxidation resulting inthe formation of N-formylkynurenine, which is a net mass increase ofabout +32 over unmodified tryptophan (FIG. 4).

A second set of experiments were performed to evaluate the percentage of2-oxo-histidines (and tryptophan dioxidation) in oligopeptides fromprotease digested FabRICATOR-cleaved aflibercept (MT4) which wasprocessed by AEX chromatography (FIG. 13 and Table 3-3 below). Thepercent of 2-oxo-histidines and tryptophan dioxidation in AEX strip foroligopeptides from protease digested FabRICATOR-cleaved aflibercept(MT4) was significantly more than the percent of 2-oxo-histidines andtryptophan dioxidation in the AEX flowthrough (referring to “MT1” inTable 3-3 below).

TABLE 3-3 Percentage of 2-oxo-Histidines Full length AEX Stripfold change Modified peptides aflibercept MT1 from MT1 AEX Strip/MT1IIW[+32]DSR (SEQ ID NO.: 28) 0.22% 0.34% 0.81% 2.4EIGLLTC[+57]EATVNGH[+14]LYK 0.00% 0.02% 0.08% 4.0 (SEQ ID NO.: 18)QTNTIIDVVLSPSH[+14]GIELSVGEK 0.01% 0.04% 0.07% 1.8 (SEQ ID NO.: 19)TELNVGIDFNWEYPSSKH[+14]QHK 0.01% 0.19% 0.42% 2.2 (SEQ ID NO.: 20)DKTH[+14]TC[+57]PPC[+57]PAPELLG  0.01%^(a) 0.11% 0.63% 5.7(SEQ ID NO.: 17) TNYLTH[+14]R (SEQ ID NO.: 21) 0.00% 0.03% 0.10% 3.3^(a)value calculated using a different peptide for full-lengthaflibercept, as the C-terminal peptide is different from MiniTrap.

The percent of 2-oxo-histidines and tryptophan dioxidation in AEX stripwas significantly more than the percent of 2-oxo-histidines andtryptophan dioxidation in the AEX flowthroughs during the MT1productions (referring to “MT1” in Table 3-3 above). Compared to Table3-2, Table 3-3 shows similar results that stripping the AEX columnproduced a sample with a significantly higher percent of2-oxo-histidines and tryptophan dioxidation compared to the percent of2-oxo-histidines and tryptophan dioxidation in AEX flowthrough,suggesting that the 2-oxo-histidines and tryptophan dioxidation speciesare bound to the AEX column during the separation and are removed uponstripping the AEX column. This is further evident in the extracted ionchromatogram as seen in FIG. 14.

Strong Cation Exchange Chromatogram (CEX)

A series of experiments were conducted in order to identify acidicspecies and other variants present in samples comprising anti-VEGFproteins.

Strong cation exchange chromatography was performed using a MonoS(10/100) GL column (GE Life Sciences, Marlborough, Mass.). For thesample separations, the mobile phases used were 20 mM2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7 (Mobile phase A) and40 mM sodium phosphate, 100 mM sodium chloride pH 9.0 (Mobile phase B).A non-linear pH gradient was used to elute charge variants of MT1 withdetection at 280 nm. Peaks that elute at a relative residence timeearlier than the main peak are designated herein as acidic species.

A sample from the MT1 lot 2 (≤BY3), prior to any enrichment, wassubjected to CEX using the method as depicted in FIG. 15. Desialylationwas applied to the sample in order to reduce the complexity of variantsof MT1. This was followed by preparative SEC processing (Superdex 200prep grade XK26/100) using 1× DPBS, pH 7.2±0.2, as the mobile phase. Thefractions obtained from the preparative SEC column comprisingdesialylated MiniTrap (dsMT1) were combined and further subjected tostrong cation exchange (SCX) chromatography to enrich for chargevariants of MT1 using a dual salt-pH gradient. The procedure resulted ina total of 7 fractions (F1-F7; MC represents the method control, FIG. 16and FIG. 17).

On performing CEX, the acidic species elute earlier than the main peaksand basic species elute after the main peaks. As observed in FIG. 17,peaks 3-5 are the main peaks. Peaks 1 and 2 are eluted before elution ofthe main species of MT1 (peaks 3-5), and thus, comprise the acidicspecies. Peak 6 is eluted after the elution of the main species of MT1(peaks 3-5), and thus, comprises the basic species. Table 3-4 shows therelative abundance of the peaks in MC (as identified in FIG. 16). Forexample, row two of Table 3-4 (labeled MC) shows that the total relativeamount of acidic species in MC is about 19.8% (i.e., peak 1+peak 2).Table-3-4 also shows the relative abundance of the peaks for eachindividual fraction. While there are overlapping species in thedifferent fractions (as reflected in FIG. 16 and FIG. 17), the majorityof fractions F1 and F2 are acidic species (i.e., peak 1 and peak 2). Forexample, fraction F1 is comprised of 63.7% peak 1 and 19.2% peak 2 (fora total of 82.9% acidic species). Fraction F2 is comprised of 9.6% peak1 and 75.9% peak 2 (for a total of 85.5% acidic species). The majorityof fractions F3-F5 are the main species of MT1 (peaks 3-5). Lastly, themajority of fractions F6-F7 are the basic species (peak 6) but doinclude some portions of the main species (e.g., peaks 4 and 5).

It was also observed that fractions F1 and F2 (which comprises theacidic species) had an intense yellow-brown coloration compared to thefractions F3-F5 (which comprises the main species or “MT1”). All thefractions were inspected for color at concentrations>13 mg/mL. Asevident from this Example, the presence of acidic species in the sampletracked with the appearance of yellow-brown coloration, removal (orminimization) of which can be accomplished by removing (or minimizing)the acidic species from MT1.

TABLE 3-4 Relative abundance of peaks based on analytical CEX Peak Area(%) Sample Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 MC  5.9 13.9 15.025.4 20.9 19.0 MT1 F1 63.7 19.2 17.1 ND ND ND MT1 F2  9.6 75.9 10.6  2.2 1.6 ND MT1 F3 ND  5.0 57.2 37.8 ND ND MT1 F4 ND ND 16.3 56.3 27.4 NDMT1 F5 ND ND ND 33.1 50.4 16.5 MT1 F6 ND ND ND 16.0 27.7 56.3 MT1 F7 2.8  7.7  8.0 16.1 16.5 48.9 ND: Not Detected

The 3D chromatograms for MT1 lot 2 and fractions F1-F7 are shown inFIGS. 18A-H. MT1 lot 2 did not exhibit any significant spectral features(FIG. 18A). Fractions 1 and 2 (comprising the acidic species) exhibiteda spectral signature between 320-360 nm (see the circle in FIG. 18B).This feature was more prominent in fraction 1 compared to fraction 2(FIGS. 18B and 18C) and was absent in fraction 3 and fractions 4-7 (mainspecies, MT1) (FIGS. 18D and 18H), which did not exhibit yellow-browncoloration.

Thus, as observed above, CEX led to identification of acidicspecies/acidic fractions (fractions 1 and 2) which show an intenseyellow-brown coloration as compared to the main species/fractions(fractions 3-6). This result was also observed in the form of a distinctspectral signature present in the 3D chromatograms of fractions F1-F2and absent in fractions F3-F7.

Imaged Capillary Isoelectric Focusing (icIEF) Electropherograms

The distribution of variants in fractions F1-F7 and MC (from MT1-lot 2after CEX) was further assessed by icIEF (FIG. 19).

The distribution of variants in fractions F1-F7 and MC (from MT1-lot 2after CEX) was further assessed by icIEF using an iCE3 analyzer(ProteinSimple) with a fluorocarbon coated capillary cartridge (100 μm×5cm). The ampholyte solution consisted of a mixture of 0.35% methylcellulose (MC), 0.75% Pharmalyte 3-10 carrier ampholytes, 4.2%Pharmalyte 8-10.5 carrier ampholytes, and 0.2% pI marker 7.40 and 0.15%pI marker 9.77 in purified water. The anolyte was 80 mM phosphoric acid,and the catholyte was 100 mM sodium hydroxide, both in 0.10%methylcellulose. Samples were diluted in purified water and sialidase Awas added to each diluted sample at an enzyme to substrate ratio of1:200 (units of sialidase A per milligram of MT1) followed by incubationat ambient temperature for approximately 16 hours. The sialidase Atreated samples were mixed with the ampholyte solution and then focusedby introducing a potential of 1500 V for one minute followed by apotential of 3000 V for 7 minutes. An image of the focused MT1 variantswas obtained by passing 280 nm ultraviolet light through the capillaryand into the lens of a charge coupled device digital camera. This imagewas then analyzed to determine the distribution of the various chargevariants (FIG. 19). Referring to FIG. 19, fractions F1 and F2 (or theacidic fractions) showed an absence of the peak for MT1, which isclearly observed for MC and fractions F3-F7 (main species, MT1). Thus,icIEF electropherograms were considered able to detect and determine thedistribution of the different charge variants of the protein underconsideration, MT1 in this case. Thus, it was evident that acidicfractions on performing CEX analysis showed (a) increased relativeabundance of percent of 2-oxo-histidine or dioxo-tryptophan (Table 3-2(II)); (b) increased yellow-brown coloration (data not shown); and (c)presence of a spectral signature as seen on the 3D chromatograms forfractions 1 and 2 (FIG. 18B and FIG. 18C).

Example 4 Photo-Induction Study

In this Example, photo-induction of VEGF MiniTrap (MT), for example MT1,was performed by exposure of a protein sample to varying amounts of coolwhite (CW) fluorescent light or ultra-violet A (UVA) light. The colorand oxidized amino acid content of the light exposed samples wasdetermined. LCMS analysis was performed following exposure, as explainedabove. Exposure of MT to cool white light or UVA light produced anincrease in oxidized amino acid residues, for example, histidine (Table4-1, Table 4-2 and Table 4-3).

TABLE 4-1 Photo-Induction Study Design 0.2 0.5 0.8 1.0 2.0 CumulativeExposure (×ICH) (×ICH) (×ICH) (×ICH)H (×ICH) CW fluorescent exposure0.24 million 0.6 million 0.96 million 1.2 million 2.4 million (lux*hr)lux*hr lux*hr lux*hr lux*hr lux*hr Incubation time 30 hours 75 hours 100hours 150 hours 300 hours with CW fluorescent light (at 8 klux) UVAexposure 40 100 160 200 400 (W*hr/m²) Incubation time with UVA 4 hours10 hours 16 hours 20 hours 40 hours (at 10 W/m²) ICH refers to ICHHarmonised Tripartite Guideline: Stability Testing: PhotostabilityTesting of New Drug Substances and Products Q1B which specifiesphotostability studies to be conducted with not less than 1.2 millionlux*hours cool white fluorescent light and near ultraviolet energy ofnot less than 200 W*hr/m².

Table 4-2 depicts the increase in coloration of the MT sample exposed tocool-white light and ultra-violet light. For example, b-value for sample(t=0) was 9.58. On exposing this sample to cool-white light at 2.4million lux*hr, the b-value increases to 22.14. This increase in b-valueindicates that the exposure of MT to cool-white light at 2.4 millionlux*hr increases yellow-brown coloration of the sample as compared tosample (t=0). Similarly, on exposing MT sample (t=0) to ultra-violetlight at 400 W*h/m², the b-value increases to 10.72 from 9.58. Thisincrease in b-value indicates that the exposure of MT sample toultra-violet light at 400 W*h/m² produces an increased yellow-browncoloration of the sample as compared to sample (t=0).

TABLE 4-2 Color of Samples Exposed to Cool White Light and Ultra-VioletLight Photo exposure ×ICH (lux*hr) L* a* b* BY Value Cool White Light T= 0 97.37 −1.12 9.58 4.0 0.2× (0.24 million lux*hr) 96.46 −0.72 11.753.7 0.5× (0.6 million lux*hr) 95.47 −0.4 11.3 3.7 0.8× (0.96 millionlux*hr) 95.33 −0.38 11.96 3.6 l.0× (1.2 million lux*hr) 94.42 −0.2 13.723.3 2.0× (2.4 million lux*hr) 92.70 0.41 22.14 2.0 UVA 0.2× (40 W*h/m²)97.26 −0.92 12.66 3.5 0.5× (100 W*h/m²) 100.39 −1.01 11.83 3.7 0.8× (160W*h/m²) 79.69 −0.18 10.1 3.6 l.0× (200 W*h/m²) 97.48 −0.95 11.36 3.72.0× (400 W*h/m²) 97.76 −0.98 10.72 3.8 Sample colors are indicatedusing the CIELAB color space (L*, a* and b* variables) and relative tothe EP BY color standard; L* = white to black (L* is lightness); a* =magenta to aqua; b* = yellow to blue, the higher the b-value the moreyellow.

TABLE 4-3 (I)2-oxo-His Levels in Peptides from Ultra-Violet Light Stressed MiniTrapPeptides Site t0 UV_4 h UV_10 h UV_16 h UV_20 h UV_40 h DKTHTCPPCPAPELH209 0.056% 0.067% 0.081% 0.088% 0.077% 0.091% LG (SEQ ID NO.: 17)EIGLLTCEATVNGH H86 0.010% 0.020% 0.034% 0.037% 0.033% 0.035% LYK (SEQ IDNO.: 18) QTNTIIDVVLSPSH H110 0.024% 0.031% 0.028% 0.028% 0.027% 0.027%GIELSVGEK (SEQ ID NO.: 19) TELNVGIDFNWEYP H145 0.096% 0.147% 0.163%0.173% 0.147% 0.125% SSKHQHK (SEQ ID NO.: 20) TNYLTHR (SEQ ID H95 0.014%0.032% 0.044% 0.056% 0.058% 0.078% NO: 21) SDTGRPFVEMYSEI H19 0.007%0.013% 0.021% 0.025% 0.024% 0.034% PEIIHMTEGR (SEQ ID NO.: 22)VHEKDK (SEQ ID H203 0.040% 0.105% 0.238% 0.255% 0.269% 0.324% NO.: 23)

TABLE 4-3 (II)2-oxo-His Levels in Peptides from Cool White Light Stressed MiniTrapPeptides Site t0 CW_30 h CW_75 h CW_100 h CW_150 h CW_300 hDKTHTCPPCPAPELL H209 0.056% 0.152% 0.220% 0.243% 0.258% 0.399%G (SEQ ID NO.: 17) EIGLLTCEATVNGHL H86 0.010% 0.063% 0.110% 0.132%0.170% 0.308% YK (SEQ ID NO.: 18) QTNTIIDVVLSPSHGI H110 0.024% 0.085%0.120% 0.128% 0.148% 0.180% ELSVGEK (SEQ ID  NO.: 19) TELNVGIDFNWEYPH145 0.096% 0.423% 0.585% 0.634% 0.697% 0.748% SSKHQHK (SEQ ID  NO.: 20)TNYLTHR (SEQ ID H95 0.014% 0.103% 0.175% 0.198% 0.267% 0.437% NO.: 21)SDTGRPFVEMYSEIP H19 0.007% 0.025% 0.043% 0.049% 0.058% 0.115%EIIHMTEGR (SEQ ID NO.: 22) VHEKDK (SEQ ID H203 0.040% 0.426% 0.542%0.622% 0.702% 1.309% NO.: 23)

Exposure of aflibercept MT to cool white light or UVA light tracked withthe appearance of oxidized histidines (2-oxo-his) (Table 4-3). Referringto Table 4-3, the peptide “SDTGRPFVEMYSEIPEIIHMTEGR (SEQ ID NO.: 22)”with oxo-histidine was 0.007% in MT sample (t=0), whereas its abundanceincreased to 0.324% on exposure to ultra-violet light for 40 hours(Table 4-3(I)) and to 1.309% on exposure to cool-white light for 300hours (Table 4-3(II)).

Two species of 2-oxo-histidine were observed, a 13.98 Da species (asshown in FIG. 2) and a 15.99 Da species (as shown in FIG. 3), with the13.98 Da species being predominant in light stressed MiniTrap samples.The 15.99 Da species is known to be a product of a coppermetal-catalyzed process (Schöneich, J. Pharm. Biomed Anal., 21:1093-1097(2000)). Moreover, the 13.98 Da species is a product of a light-drivenprocess (Liu et al., Anal. Chem., 86, 10, 4940-4948 (2014)).

Similar to the increased abundance of oxidized histidines in samplesexposed to cool white light and UVA light, exposure of MT to cool whitelight or UVA light also induced formation of other PTMs (Table 4-4 andTable 4-5).

TABLE 4-4 (I)Other PTMs in Peptides from Ultra-Violet Light Stressed MiniTrapPeptides Site t0 UV_4 h UV_10 h UV_16 h UV_20 h UV_40 h DeamidationEIGLLTCEATVNGHLYK (SEQ N84 20.8% 21.7% 21.5% 21.5% 22.7% 22.4%ID NO.: 62) QTNTIIDVVLSPSHGIELSVGE N99  5.3%  5.4%  5.5%  5.4%  5.5% 5.6% K (SEQ ID NO.: 63) Oxidation SDTGRPFVEMYSEIPEIIHMTE M10  4.5% 8.2% 11.1% 13.3% 13.8% 19.3% GR (SEQ ID NO.: 64) SDTGRPFVEMYSEIPEIIHMTEM20  1.1%  2.0%  2.8%  3.4%  3.4%  4.6% GR (SEQ ID NO.: 65)TQSGSEMK (SEQ ID NO.: 66) M163  2.0%  2.7%  4.1%  4.6%  7.9%  8.7%SDQGLYTCAASSGLMTK M192  5.4%  8.1% 10.9% 12.1% 12.5% 18.3%(SEQ ID NO.: 67) 3-deoxygluconasone SDTGRPFVEMYSEIPEIIHMTE R5  9.9%10.0%  9.9%  9.7%  9.8%  9.3% GR (SEQ ID NO.: 68)

TABLE 4-4 (II)Other PTMs in Peptides from Cool White Light Stressed MiniTrap PeptidesSite t0 CW_30 h CW_75 h CW_100 h CW_150 h CW_300 h DeamidationEIGLLTCEATVNGHLYK (SEQ N84 20.8% 22.0% 22.9% 20.3% 21.8% 21.3%ID NO.: 62) QTNTIIDVVLSPSHGIELSVGEK N99  5.3%  5.6%  5.2%  5.6%  5.5% 5.8% (SEQ ID NO.: 63) Oxidation SDTGRPFVEMYSEIPEIIHMTEG M10  4.5% 11.7%17.3% 19.9% 25.1% 39.7% R (SEQ ID NO.: 64) SDTGRPFVEMYSEIPEIIHMTEG M20 1.1%  3.1%  4.3%  5.1%  6.1%  8.2% R (SEQ ID NO.: 65)TQSGSEMK (SEQ ID NO.: 66) M163  2.0%  3.3% 15.7% 11.7% 26.4% 20.5%SDQGLYTCAASSGLMTK (SEQ M192  5.4% 10.7% 15.3% 18.7% 22.8% 37.6%ID NO.: 67) 3-deoxygluconasone SDTGRPFVEMYSEIPEIIHMTEG R5  9.9%  9.9% 9.6%  9.3%  9.3%  9.0% R (SEQ ID NO.: 68)

TABLE 4-5 (I)Oxidation Levels of Tryptophan/Tyrosine/Phenylalanine in Peptides fromUltra-Violet Light Stressed MiniTrap Modifica- Peptides tion Site t0UV_4 h UV_10 h UV16 h UV_20 h UV_40 h SDQGLYTCAASSGLM  +4 W58 0.016%0.049% 0.089% 0.119% 0.132% 0.221% TK (SEQ ID NO.: 67) +16 0.047% 0.109%0.177% 0.225% 0.242% 0.514% +32 0.200% 0.487% 0.415% 0.481% 0.423%0.498% +48 0.000% 0.000% 0.000% 0.001% 0.001% 0.001% TELNVGIDFNWEYPSS +4 W138 0.435% 0.462% 0.550% 0.557% 0.502% 0.512% K (SEQ ID NO.: 29)+16 0.083% 0.100% 0.161% 0.206% 0.239% 0.448% +32 0.009% 0.018% 0.027%0.039% 0.044% 0.115% +48 0.284% 0.278% 0.270% 0.302% 0.343% 0.275%GFIISNATYK (SEQ ID +16 Y64 0.032% 0.041% 0.046% 0.053% 0.054% 0.073%NO.: 69) KFPLDTLIPDGK (SEQ +16 F44 0.068% 0.077% 0.087% 0.084% 0.070%0.096% ID NO.: 70) FLSTLTIDGVTR (SEQ +16 F166 0.066% 0.075% 0.085%0.089% 0.089% 0.124% ID NO.: 71)

TABLE 4-5 (II)Oxidation Levels of Tryptophan/Tyrosine/Phenylalanine in Peptides fromCool White Light Stressed MiniTrap Modifica- Peptides tion Site t0CW_30 h CW_75 h CW_100 h CW_150 h CW_300 h SDQGLYTCAASSG  +4 W58 0.016%0.063% 0.124% 0.161% 0.228% 0.526% LMTK (SEQ ID NO.: +16 0.047% 0.129%0.227% 0.283% 0.377% 0.795% 67) +32 0.200% 1.601% 2.706% 3.139% 3.925%6.974% +48 0.000% 0.001% 0.002% 0.002% 0.003% 0.005% TELNVGIDFNWEY  +4W138 0.435% 0.555% 0.481% 0.490% 0.429% 0.522% PSSK (SEQ ID NO.: +160.083% 0.109% 0.364% 0.251% 0.399% 0.753% 29) +32 0.009% 0.019% 0.027%0.033% 0.048% 0.135% +48 0.284% 0.284% 0.330% 0.308% 0.347% 0.316%GFIISNATYK (SEQ +16 Y64 0.032% 0.043% 0.057% 0.063% 0.078% 0.127%ID NO.: 69) KFPLDTLIPDGK +16 F44 0.068% 0.087% 0.072% 0.088% 0.079%0.144% (SEQ ID NO.: 70) FLSTLTIDGVTR +16 F166 0.066% 0.091% 0.088%0.101% 0.112% 0.168% (SEQ ID NO.: 71)

Thus, exposure of MT to cool white light or UVA light tracked with theappearance of oxidized residues (such as histidines/tryptophans(oxo-Trp)). Four species of oxo-trp were observed: +4 Da, +16 Da, +32 Daand +48 Da. The +4 Da species is explained by formation of kynurenine(FIG. 4), whereas the +16 Da, +32 Da and +48 Da are the mono-oxidation,di-oxidation and tri-oxidation of tryptophan residues. Peptide mappingof tryptic digests of MT samples monitored at 320 nm is shown in FIG.20. The relative presence of oxidized residues comprising peptides canbe compared in FIG. 20. For example, for the peptide IIW(+4)DSRK (SEQ IDNO.: 114), a significant difference in its presence can be seen for MTsample at t=0, and MT1 sample exposed to UVA for 40 hour and MT sampleexposed to CWL for 300 hours.

Exposure of MT to cool white light or UVA light was also evaluated forthe presence of BMW/low molecular weight (LMW) species (Table 4-6).

TABLE 4-6 HMW/LMW Species Were Generated After Extended UVA and CWLStress Sample: MT1, 80 mg/mL, pH 5.8 Light Dark Light Dark Light Darkexposed control exposed control exposed control Samples samples Samplessamples Samples samples Cumulative UVA exposure (× ICH) % HMW % Native %LMW t = 0  2.1 NA 96.7 NA 1.2 NA 0.2 × ICH (40  2.1 2.1 96.7 96.7 1.21.3 W*h/m2) 0.5 × ICH (100 11.6 2.2 86.5 96.6 1.9 1.2 W*h/m2) 0.8 × ICH(160 14.5 2.2 83.4 96.6 2.1 1.2 W*h/m2) 1.0 × ICH (200 15.8 2.2 81.996.6 2.3 1.3 W*h/m2) 2.0 × ICH (400 22.7 2.3 74.5 96.7 2.8 1.0 W*h/m2)Cumulative CWL exposure (× ICH) % HMW % Native % LMW 0.2 × ICH (0.2412.1 2.2 86.6 96.6 1.4 1.2 million lux* h) 0.5 × ICH (0.6 20.4 2.3 77.996.4 1.6 1.3 million lux*hr) 0.8 × ICH 23.2 2.4 75.1 96.2 1.7 1.4 (0.96million lux*hr) 1.0 × ICH (1.2 30.1 2.6 68.1 96.2 1.9 1.3 millionlux*hr) 2.0 × ICH (2.4 45.0 2.9 52.6 95.8 2.4 1.4 million lux*hr)

To track the coloration with respect to HMW/LMW species for each sample,analytical size-exclusion chromatography with full-spectrum PDAdetection (SEC-PDA) was performed as shown above on all the stressedsamples (CWL and UVA). SEC-PDA analysis of CWL-stressed MT revealssignificant increases in absorbance at ˜350 nm for all size variantsexcept the LMW species (FIG. 21), whereas SEC-PDA on UVA-stressed MTreveals no increases in absorbance at ˜350 nm (FIG. 22). UnlikeCWL-treated stress samples, UVA-treated stress samples did not produceany significantly quantifiable yellow-brown color.

A similar result was obtained after studying absorbance ratios at 320 nmand 280 nm for the samples stressed by UVA and CWL. The A320/A280ratios, analyzed by either raw intensity or total peak area, trackedwith increasing intensity of yellow color in CWL-stressed samples (FIG.23), whereas the A320/A280 ratios did not track with increasingintensity of yellow color in UVA-stressed samples (FIG. 24). Thiscorroborates the previous observation that MT1 samples subjected to UVAstress does not result in the same yellow-brown color observed followingCWL stress.

Example 5 Upstream Methods for Reducing Coloration 5.1 ChemicallyDefined Medium Incubation Study

The effect of various constituents spiked into fresh chemically definedmedia (CDM) comprising aflibercept with respect to coloration wasexamined.

One or more 50 mL vent-capped shaker tubes with 10 mL working volume(fresh CDM1) were incubated for 7 days, taking samples on day 0 and day7. Aflibercept samples (aflibercept recombinant protein in an aqueousbuffered solution, pH 6.2, comprising 5 mM sodium phosphate, 5 mM sodiumcitrate and 100 mM sodium chloride) were spiked into shaker tubes at aconcentration of 6 g/L.

Components added to reach a cumulative concentration:

-   -   Cysteine: 16.6 mM    -   Iron: 0.23 mM    -   Copper: 0.0071 mM    -   Zinc: 0.54 mM

The scaled effect of each constituent added on the b* value (CIE L*, a*,b* color space) is set forth in FIG. 25A and plot of actual b* valueagainst predicted b* value is set forth in FIG. 25B. Addition ofcysteine resulted in the largest yellow-brown color increase. Iron andzinc also generated color. Folic acid and B vitamin group (includingthiamine, niacinamide, D-pantothenic acid, D-biotin, and pyridoxine)increased the yellow-brown color. Riboflavin and Vitamin B12 did notstatistically impact color.

5.2 Effect of Decreasing Cysteine and Metals on b* Value

Bioreactors (e.g., 2L) were inoculated from a seed culture of anaflibercept producing cell line. The inoculated cultures were grown at atemperature of 35.5° C., pH of 7.1±0.25, and air sparge set point of 22ccm. Glucose, antifoam, and basal feeds were supplemented to thebioreactors as needed. The effect of lowering the concentration ofcysteine and of metals on color when aflibercept is expressed wasevaluated in CDM1.

-   Medium at day 0=CDM1, including 1.48 mM of cysteine    -   Nutrient Feeds:        -   Day 2=Chemically defined feed (CDF)+1.3-2.1 mM cysteine        -   Day 4=CDF+1.6-1.7 mM cysteine        -   Day 6=CDF+1.6-1.7 mM cysteine        -   Day 8=CDF+1.6-1.7 mM cysteine

The bioreactor conditions were as follows:

-   -   Cysteine was added at a cumulative concentration of about 6-7        millimoles per L of culture, 8-9 millimoles per L of culture, or        10-11 millimoles per L of culture.    -   Metals in CDM1 (0.5×, 1×, or 1.5× CDM1 levels) at 1× levels are        listed below (where the concentrations are prior to inoculum        addition):        -   Fe=68-83 micromoles per liter of culture        -   Zn=6-7 micromoles per liter of culture        -   Cu=0.1-0.2 micromoles per liter of culture        -   Ni=0.5-1 micromoles per liter of culture            Decreasing cumulative cysteine levels to 6-7 millimoles/L            reduced yellow-brown color with no significant impact to            titer. Decreasing metal concentrations to 0.5× in the medium            reduced color with significant increase in titer. There was            a minimal impact to titer, VCC (viable cell concentration),            viability, ammonia or osmolality (See FIG. 26A-E). The            predicted scale effect of metal content and cysteine on b*            value and titer is set forth in FIG. 27.

5.3 Evaluation of the Effect of Antioxidants on b* Value

The effect of the antioxidants taurine, hypotaurine, thioctic acid,glutathione, glycine and vitamin C on color when spiked into spent CDMcomprising aflibercept was evaluated. One or more 50 mL vent-cappedshaker tubes with 10 mL working volume (CDM1) were incubated for 7 days,taking samples on day 0 and day 7.

The conditions for component additions to spent CDM1 were as follows:

-   -   Aflibercept sample (aflibercept recombinant protein in an        aqueous buffered solution, pH 6.2, comprising 5 mM sodium        phosphate, 5 mM sodium citrate and 100 mM sodium chloride)        spiked into shaker tubes at 6 g/L concentration    -   Antioxidants added to spent CDM1 at the following        concentrations:        -   Taurine=10 mM of culture        -   Hypotaurine=10 mM of culture        -   Glycine=10 mM of culture        -   Thioctic Acid=0.0024 mM of culture        -   Glutathione, reduced=2 mM of culture        -   Hydrocortisone=0.0014 mM of culture        -   Vitamin C (ascorbic acid)=0.028 mM of culture

Multiple antioxidants decreased color formation in spent medium: acombination of hypotaurine, taurine and glycine; thioctic acid; andvitamin C. Glutathione increased b* value.

TABLE 5-1 Summary of Antioxidant Effect on Color Formation of MiniTrapCondition b* value Spent Medium Day 0 0.37 Spent Medium Day 7 Control1.47 Spent Medium Day 7 + Antioxidants* 1.02 * Antioxidants thatsignificantly decreased b* value: Hypotaurine/Taurine/Glycine, ThiocticAcid, Vitamin C.

A summary of the predicted effect of various antioxidants on b* value(CIE L*, a*, b* color space) is set forth in FIG. 28(A-C).

The effect of the further addition to the antioxidants on color, whenspiked into spent CDM comprising aflibercept, was evaluated. One or more50 mL vent-capped shaker tubes with 10 mL working volume (CDM1) wereincubated for 7 days, taking samples on day 0 and day 7.

The conditions for component additions to spent CDM1 were as follows:

-   -   Aflibercept sample (aflibercept recombinant protein in an        aqueous buffered solution, pH 6.2, comprising 5 mM sodium        phosphate, 5 mM sodium citrate and 100 mM sodium chloride)        spiked into shaker tubes at 6 g/L concentration    -   Two DOE experiments were run:    -   (i) Antioxidants added to spent CDM1 at the following        concentrations:        -   Taurine=10 mM of culture        -   Hypotaurine=10 mM of culture        -   Glycine=10 mM of culture        -   Thioctic Acid=0.0024 mM of culture        -   Vitamin C (ascorbic acid)=0.028 mM of culture    -   (ii) Antioxidants added to reach the following cumulative        concentrations:        -   ATA=2.5 μM-5 μM        -   Deferoxamine mesylate (DFO)=5μM-10 μM        -   Catalase=101.5 mg/L        -   S-carboxymethyl-L-Cysteine=10 mM

Hypotaurine was found to decrease the color formation in spent medium(FIG. 28D). DFO also significantly decreased the color formation inspent medium (FIG. 28D). The other antioxidants did not have astatistical impact on the color formation.

TABLE 5-2 Summary of Antioxidant Effect on Color Formation of MiniTrapCondition b* value Spent Medium Day 0 0.44 Spent Medium Day 7 Control1.73 Spent Medium Day 7 + Hypotaurine 1.32 Spent Medium Day 7 + DFO 0.92

Shake-Flask Antioxidant Study:

Taurine, hypotaurine, glycine, thioctic acid and vitamin C wereevaluated individually and in combination for their ability to decreasethe color formation in cell culture (Table 5-3).

250 mL shake flasks were inoculated from a seed culture of anaflibercept producing cell line. The inoculated cells were grown at35.5° C. in incubators with 5% CO₂ control. Glucose and basal feeds weresupplemented to the shake flasks as needed. The process described abovewas used wherein metals were present at 0.5× concentration in CDM1 andcysteine was added at a cumulative concentration of 6-7 mM.

TABLE 5-3 Level 1 Level 1 Level 1 Antioxidant 0× 0.5× 1× Taurine 0 3.75mM 7.5 mM Hypotaurine 0 3.75 mM 7.5 mM Glycine 2.0 mM 5.75 mM 9.5 mMThioctic acid 1.0 μM 1.9 μM 2.8 μM Vitamin C 0 11.0 μM 21.0 μM

FIG. 28E shows the predicted effect of the antioxidants in Table 5-3 onb* value (CIE L*, a*, b* color space) and final titer. Taurine,hypotaurine, and glycine significantly reduced b* value withoutnegatively impacting titer.

Example 6 Glycosylation and Viability Studies for Aflibercept ProductionUsing CDM

Bioreactors (e.g., 2 L) were inoculated from a seed culture of anaflibercept producing cell line. The inoculated cultures were grown at atemperature of 35.5° C., pH of 7.1±0.25, and air sparge set point of 22ccm. Glucose, antifoam, and basal feeds were supplemented to thebioreactors as needed. Production of aflibercept protein was carried outusing CDM1 (proprietary). Production of a host cell line expressingaflibercept fusion protein was carried out using CDM1 (proprietary),CDM2 (commercially obtained), and CDM3 (commercially obtained). A set ofexperiments was carried out using CDM 1, 2, and 3 with no additionalmedia components. Another set of experiments was performed using CDMs1-3 to which manganese (manganese chloride tetrahydrate, Sigma, 3.2mg/L), galactose (Sigma, 8 g/L), and uridine (Sigma, 6 g/L) were addedto the feeds to modify the galactosylation profile. Lastly, a set ofexperiments was performed using CDMs 1-3 to which manganese (manganesechloride tetrahydrate, Sigma, 3.2 mg/L), galactose (Sigma, 8 g/L), anduridine (Sigma, 6 g/L) were added to the feeds to modify thegalactosylation profile and dexamethasone (Sigma, 12 mg/L) was added tothe feeds to modify the sialyation profile of the composition. Aclarified harvest using each of the CDM was prepared by centrifugationfollowed by 0.45 μm filtration.

Samples were processed by Protein A prior to N-glycan analysis.

Titer Measurements

Throughout these examples, unless stated otherwise, aflibercept titerswere measured daily using an Agilent (Santa Clara, Calif.) 1200 SeriesHPLC, or equivalent, operating with a low pH, and step elution gradientwith detection at 280 nm. Concentrations were assigned with respect to areference standard calibration curve.

Viable Cell Density (VCD) and Cell Viability Values

Throughout these examples, unless stated otherwise, viable cell density(VCD) and cell viability values were measured through trypan blueexclusion via Nova BioProfile Flex automated cell counters (NovaBiomedical, Waltham, MA). Glucose, lactate, offline pH, dissolved oxygen(DO), pCO2 measurements, and osmolality were measured with the NovaBioProfile Flex (Nova Biomedical, Waltham, Mass.).

N-Glycan Oligosaccharide Profiling

-   -   Approximately 15 μg of Protein A processed samples from CDM 1-3        harvests were prepared for N-glycan analysis in accordance with        the Waters GlycoWorks protocol using the GlycoWorks Rapid        Deglycosylation and GlycoWorks RapiFluor-MS Label kits (Waters        part numbers 186008939 and 186008091, respectively). N-glycans        were removed from the aflibercept protein by treating the        samples with PNGase-F at 50.5° C. for 5 minutes, followed by a        cool down at 25° C. for 5 minutes. The released glycans were        labeled with RapiFluor-MS fluorescent dye through reaction at        room temperature for 5 minutes. The protein was precipitated by        adding acetonitrile to the reaction mixture and pelletized to        the bottom of the well through centrifugation at 2,204×g for 10        minutes. The supernatant comprising the labeled glycans was        collected and analyzed on an UPLC using hydrophilic interaction        liquid chromatography (Waters BEH Amide column) with post-column        fluorescence detection. After binding to the column, the labeled        glycans were separated and eluted using a binary mobile phase        gradient comprised of acetonitrile and aqueous 50 mM ammonium        formate (pH 4.4). The labeled glycans were detected using a        fluorescence detector with an excitation wavelength of 265 nm        and an emission wavelength of 425 nm. Using the relative area        percentages of the N-glycan peaks in the resultant        chromatograms, the N-glycan distribution is reported as the        total percentage of N-glycans (1) containing a core fucose        residue (Total Fucosylation, Table 6-1), (2) containing at least        one sialic acid residue (Total Sialylation, Table 6-2), (3)        identified as Mannose-5 (Mannose-5, Table 6-3), (4) containing        at least one galactose residue (Total Galactosylation, Table        6-4), and (5) of known identity (Total Identified Peaks, Table        6-5).

Results

The viable cell count (VCC), viability, and harvest titer results areshown in FIGS. 29-31 for CDMs 1-3 with and without additionalcomponents.

Amongst the nine cultures, the CDM1 culture comprising uridine,manganese, and galactose showed the highest titer at 12 days (5.5 g/L).CDM1 without additional components also showed a high titer at 12 days(about 4.25 g/L) compared to the other seven cultures (FIG. 29).

Cell viability results were similar across the various conditions up toprocess day 6. After process day 7, the CDM2 and CDM3 cultures with orwithout additional media components showed more than about 90% viability(FIG. 30).

CDM1 culture with uridine, manganese and galactose showed the highestVCC around day 6 (FIG. 31).

The influence of cultures and supplements had a significant impact onthe overall N-glycan distribution (Tables 6-1 to 6-5). The glycan levelswere compared using Protein A processed aflibercept (two samples wereevaluated) made using soy hydrolysate. The total identified peaks arelisted in Table 6-5.

TABLE 6-1 Total Fucosylation (%) Condition Day 6 Day 10 Day 12 CDM148.75 — 46.26 CDM1 + UMG 49.21 — 44.38 CDM1 + UMG + Dex 48.88 — 46.23CDM2 — 45.68 45.14 CDM2 + UMG — 46.36 45.27 CDM2 + UMG + Dex — 46.92 —CDM3 49.24 — 45.59 CDM3 + UMG 48.71 — 42.61 CDM3 + UMG + Dex 49.36 —44.56 Soy hydrolysate 1 51.37 Soy hydrolysate 2 52.43 U is uridine, M ismanganese, G is galactose, Dex is dexamethasone

TABLE 6-2 Total Sialylation (%) Condition Day 6 Day 10 Day 12 CDM1 44.06— 39.14 CDM1 + UMG 43.72 — 35.8 CDM1 + UMG + Dex 43.2 — 36.72 CDM2 —37.62 36.67 CDM2 + UMG — 37.57 36.29 CDM2 + UMG + Dex — 38.06 — CDM3 44— 31.21 CDM3 + UMG 42.48 — 30.84 CDM3 + UMG + Dex 43.82 — 32.74 Soyhydrolysate 1 58.24 Soy hydrolysate 2 59.23 U is uridine, M ismanganese, G is galactose, Dex is dexamethasone

TABLE 6-3 Mannose-5 (%) Condition Day 6 Day 10 Day 12 CDM1 6.76 — 10.1CDM1 + UMG 6.9 — 13.17 CDM1 + UMG + Dex 6.23 — 8.86 CDM2 — 9.71 11.96CDM2 + UMG — 9.44 10.93 CDM2 + UMG + Dex — 8.21 — CDM3 2.31 — 12.63CDM3 + UMG 2.71 — 13.38 CDM3 + UMG + Dex 2.05 — 11.98 Soy hydrolysate 15.19 Soy hydrolysate 2 5.24 U is uridine, M is manganese, G isgalactose, Dex is dexamethasone

TABLE 6-4 Total Galactosylation (%) Condition Day 6 Day 10 Day 12 CDM168.44 — 62.9 CDM1 + UMG 69.25 — 59.02 CDM1 + UMG + Dex 69.05 — 63.26CDM2 — 65.33 63.68 CDM2 + UMG — 68.13 66 CDM2 + UMG + Dex — 69.35 — CDM374.57 — 62.28 CDM3 + UMG 74.82 — 62.2 CDM3 + UMG + Dex 76.48 — 65.18 Soyhydrolysate 1 79.64 Soy hydrolysate 2 80.55 U is uridine, M ismanganese, G is galactose, Dex is dexamethasone

TABLE 6-5 Total Identified Peaks (%) Condition Day 6 Day 10 Day 12 CDM187.28 — 84.67 CDM1 + UMG 88.43 — 83.82 CDM1 + UMG + Dex 87.36 — 83.44CDM2 — 86.23 86.67 CDM2 + UMG — 87.81 86.87 CDM2 + UMG + Dex — 87.53 —CDM3 86.38 — 86.31 CDM3 + UMG 87.07 — 86.13 CDM3 + UMG + Dex 87.18 —87.43 Soy hydrolysate 1 93.93 Soy hydrolysate 2 94.74 U is uridine, M ismanganese, G is galactose, Dex is dexamethasone

The total fucosylation, total sialylation, total galactosylation andmannose-5 observed on day 12 of the cultures of the various CDMs was42.61% to 46.26%, 30.84% to 39.14%, 59.02 to 66% and 8.86% to 13.38%,respectively. These values for glycosylation differ from theglycosylation values obtained using soy hydrolysate.

Lastly, color measurements were carried out for the clarified harvestsobtained from cells expressing aflibercept in CDM1, CDM2, and CDM3supplemented with uridine, manganese, and galactose. The operatingparameters for the bioreactor study steps will be known to one ofordinary skill in the art.

Example 7 Affinity Production of Anti-VEGF Proteins 7.1 Expression ofVEGF MiniTrap

The coding regions of recombinant VEGF MiniTrap (e.g., MT5, SEQ ID NO.:46) were operably linked to a signal sequence, cloned into a mammalianexpression vector and transfected into Chinese hamster ovary (CHO-K1)cells; the stably transfected pools were isolated after selection with400 μg/mL hygromycin for 12 days. The stable CHO cell pools, grown inchemically defined protein-free medium, were used to produce proteinsfor testing. The recombinant polypeptides were secreted from the cellsinto the growth medium.

Sequences of constituent domains of the VEGF MiniTrap

-   -   Human Flt1 (accession #NP_001153392.1)    -   Human Flk1 (accession #NP_002244.1)    -   Human Fc (IGHG1, accession #P01857-1)

The recombinant VEGF MiniTrap (MT5) was obtained from this process andwas further processed.

7.2 Preparation of Affinity Chromatography Columns

Five distinct proteins capable of binding to the VEGF MiniTrap (MT5)were evaluated. The proteins used include VEGF165 (SEQ ID NO.: 72), mAb1(a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 73 is a heavychain and SEQ ID NO.: 74 is a light chain); mAb2 (a mouse anti-VEGFR1mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain and SEQ ID NO.: 76is a light chain); mAb3 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ IDNO.: 77 is a heavy chain and SEQ ID NO.: 78 is a light chain) and mAb4(a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 79 is a heavychain and SEQ ID NO.: 80 is a light chain).

The column was activated by washing the columns with 6 column volumes(CV) of 1 mM ice-cold hydrochloric acid at a flow rate not exceeding 1mL/min. Ten mg of each of the proteins were loaded onto three HiTrapNETS-Activated HP affinity columns (1 mL, GE Healthcare, Cat#17-0716-01) and the columns were closed to allow coupling to take placefor 30 minutes at room temperature. The columns were washed with 18column volumes of 0.5 M sodium acetate, 0.5 M NaCl, pH 4.0 and the opensites were blocked with 18 column volumes of 0.5 M Tris-HCl, 0.5 M NaClpH 8.3 (the wash was carried out in the following order: 6 columnvolumes of 0.5 M Tris-HCl, 0.5 M NaCl, pH 8.3; 6 column volumes of 0.5 Msodium acetate (sodium acetate: JT Baker, Cat #3470-01), 0.5 M NaCl, pH4.0; 6 column volumes of 0.5 M Tris, pH 8.3; incubate the column for 30minutes at room temperature; 6 column volumes of 0.5 M sodium acetatebuffer, 0.5 M NaCl pH 4.0; 6 column volumes of 0.5 M Tris-HCl, 0.5 NaCl,pH 8.3 and 6 column volumes of 0.5 M sodium acetate buffer, 0.5 M NaClpH 4.0). The columns were stored in DPBS, pH 7.5. The five columnsevaluated are designated as column 1 (comprising VEGF₁₆₅), column 2(comprising mAb1), column 3 (comprising mAb2), column 4 (comprisingmAb3) and column 5 (comprising mAb4).

7.3 Production of MiniTrap Using Affinity Chromatography

Sample Preparation. Two different production processes for the MiniTrapwere performed. In one case, material comprising a MiniTrap sample wasproduced using each of the affinity columns where the parent material(MiniTrap) was diluted in 1× DPBS buffer to 20 mg/mL and was applied tothe column and included at RT for 30 minutes. Using the affinity column,the MiniTrap was isolated from 7000 ppm of HCP.

Alternatively, harvested culture supernatant was used which comprised0.4 mg/mL of protein in the supernatant and loaded onto the differentaffinity columns (1-5) separately. No further dilution was performed.The affinity columns were then washed with 9 CV of 1× DPBS bufferfollowed by eluting the proteins with IgG elution buffer, pH 2.8(Thermo, Cat #21009).

MiniTrap material obtained as described above was then filtered througha 0.45 μm filter or centrifuged before loading onto the columns preparedas described in Section 7.2 above. Twenty-five mL of loading solutioncomprising approximately 0.4 mg/mL protein was loaded onto each of thecolumns and incubated for 20 minutes. Each column was washed with 9 CVof DPBS (Invitrogen, Cat #14190-144) before elution for equilibration.The amount of MT5 in the wash fractions is shown in Table 7-1. Thewashes were followed by elution using 6 CV of pH 2.8 (Commercial ElutionBuffer, (Thermo, Cat #21009)) and 100 mM glycine buffer pH 2.5 andfractions were quickly neutralized with the addition of 1 M Tris, pH 7.5(Invitrogen, Cat #15567-027). The amount of MiniTrap in the elutedfractions is also shown in Table 7-1.

The MiniTrap (MT5) was successfully produced from all five affinitycolumns. The yield from the column with VEGF165 was higher than comparedto mAb1 and mAb2 columns. The mAb3 and mAb4 comprising humanizedanti-VEGFR1 mAb also showed successful production of MT5 with similaryield to mAb1 and mAb2. In Table 7-1, the expected yield was calculatedbased on 100% conjugation efficiency and 1:1 molar ratio ofaffinity-captured protein to MT5.

TABLE 7-1 Column 1 Column 2 Column 3 Column 4 Column 5 Affinity ColumnVEGF₁₆₅ mAb1 mAb2 mAb3 mAb4 Conjugation Amount (mg) 10 10 10 10 10 Load(mg) 21.2 21.2 21.2 20.1 20.1 Expected (mg) ~12 ~3.2 ~3.2 ~3.2 ~3.2 Wash(mg) 14.9 13.2 12.6 15.2 14.7 Eluate (mg) 4.8 1.6 1.8 1.5 1.6

7.4 Column Stability Study

Multiple runs were carried out using columns 1 and 2 following themethod discussed in Section 7.3 (Table 7-2 for column 1 and Table 7-3for column 2).

TABLE 7-2 Production Yield Run # 1 2 3 4 5 6 7 Load (mg) 21.2 19.7 19.719.7 19.6 19.6 19.6 Wash (mg) 14.9 13.5 15.0 15.0 14.6 14.6 14.3 Eluate(mg) 4.8 5.4 5.2 4.8 5.2 4.6 4.8

TABLE 7-3 Production Yield Run # 1 2 3 4 5 6 7 Load (mg) 21.2 19.7 19.719.7 19.6 19.6 19.6 Wash (mg) 13.2 16.0 16.4 16.4 17.1 17.2 17.2 Eluate(mg) 1.6 1.8 1.8 1.8 2.0 2.0 2.0

The columns were stored at 4° C. for about 5 weeks. A similar amount ofMT5 was eluted from each production demonstrating good column stability.

7.5 Stability Study of the Produced VEGF MiniTrap

SDS-PAGE analysis of the eluted fractions from the three columns (column1, column 2, and column 3) was performed. The samples were prepared innon-reducing and reducing SDS-PAGE sample buffer and run on a 4-12%gradient NuPage bis-Tris gel using 1× MES (Cat. No. NP0322, Invitrogen,Carlsbad, Calif.).

The wells were loaded with (1) molecular weight standard, (2) loadingsolution, (3) column wash from column 1, (4) eluted fraction from column2, (5) eluted fraction from column 1, (6) eluted fraction from column 3,(7) MT5 stored at pH 2.8 for 1 min, (8) MT5 stored at pH 2.8 for 30 min,and (9) molecular weight standard (FIG. 33 and FIG. 34). The analysisdemonstrated that fractions obtained from the eluted fractions from allthe three affinity columns (columns 1-3) showed similar size profilesand the use of the affinity columns did not destabilize the MiniTrap.

7.6 Host Cell Protein Level Calculations

A standard curve of concentration of host cell proteins was obtainedusing CHO HCP ELISA Kit, 3G (F550) (Cygus Technologies) (FIG. 32 andTable 7-4). The amount of HCPs in the loading solutions and the elutedfractions was calculated using the standard curve as depicted in FIG. 32and curve formula listed in Table 7-4.

TABLE 7-4 Low EC₅₀ High Curve Formula Asymptote Slope (ng/mL) AsymptoteR² Y = (A − D)/ 0.2 1.9 32.9 2.3 1 (1 + (X/C){circumflex over ( )}B) + D

The total HCPs were calculated using the standard curve and the chartwith the total amount of host cell proteins is shown in FIG. 35A. FIG.35B also shows total amount of host cell proteins in the load comparedto the washes and eluted fractions from columns 1, 2, 4 and 5. Multipleruns were carried out using the columns and the (#) in FIG. 35Brepresent the run from which the fraction was evaluated.

The use of affinity capture using proteins capable of binding toMiniTrap showed an efficient reduction of HCPs from about 7000 ppm toabout 25-50 ppm. As observed for the yield, the column with VEGF165showed higher purity of MiniTrap from HCPs than shown by mAb1 and mAb2columns.

7.7 SEC Profiles of VEGF MiniTrap Before and After Affinity Production

SEC profiles of the eluted fractions from three columns (columns 1-3)were compared to the SEC profile of MiniTrap in the loading solution. Asseen in FIG. 36 and Table 7-5, the SEC profiles of the MT5 before orafter affinity production were highly similar.

TABLE 7-5 Peak Retention Peak % Retention Peak % Retention Peak %Retention Peak % No. as Time Area Time Area Time Area Time Area inFIG.36 Loading solution Column 1 Eluate Column 2 Eluate Column 3 Eluate1 6.8 1.8 6.8 1.2 6.9 1.1 7.0 1.2 2 7.8 94.6 7.9 97.2 7.9 97.3 7.9 98.33 9.4 3.6 10.2 1.7 11.4 1.6 11.3 0.57.8 Kinetics of VEGF MiniTrap Pre and Post Column Samples Binding tomAb1, mAb2 and VEGF₁₆₅

Kinetic studies were performed using a Biacore T200 instrument.

Equilibrium dissociation constants (KD values) for VEGF165 binding toMiniTrap in the eluates from columns 1 and 2 and loading solution weredetermined using a real-time surface plasmon resonance biosensor using aBiacore T200 instrument. All binding studies were performed in 10 mMHEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20, pH 7.4(HBS-ET) running buffer at 25° C. The Biacore sensor surface was firstderivitized by amine coupling with a mAb 1 to capture MT5. A cartoonrepresentation of the binding study is shown in FIG. 37.

Briefly, the eluates from the columns and loading solution were dilutedinto an HBS-EP (Biacore) buffer and injected across the immobilizedprotein matrices at a capture level of ˜70 RUs. The VEGF₁₆₅ was theninjected at a flow rate of 50 μL/min. Equivalent concentration ofanalyte was simultaneously injected over an untreated reference surfaceto serve as blank sensorgrams for subtraction of bulk refractive indexbackground. The sensor chip surface was regenerated between cycles withtwo 5-min injections of 10 mM Glycine, at 25 μL/min. The resultantexperimental binding sensorgrams were then evaluated using the BIAevaluation 4.0.1 software to determine kinetic rate parameters. Datasetsfor each sample were fit to the 1:1 Langmuir model. For these studies,binding and dissociation data were analyzed under the Global FitAnalysis protocol while selecting fit locally for maximum analytebinding capacity (RU) or Rmax attribute. In this case, the softwarecalculated a single dissociation constant (kd), association constant(ka), and affinity constant (Kd). The equilibrium dissociation constantis K_(D)=kd/ka. The kinetic on-rate, the kinetic off rate, and theoverall affinities were determined by using different VEGF₁₆₅concentrations in the range of 0.03-2 nM (Table 7-6). The dissociativehalf-lives (t^(1/2)) were calculated from the kinetic rate constants as:t_(1/2)=ln(2)/60*Kd. Binding kinetic parameters for MT5 to VEGF₁₆₅obtained from before and after the affinity chromatography production at25° C. are shown in Table 7-6.

The affinity (KD), on rate (ka, M−1s−1) and off rate (kd) for MT5produced by affinity chromatography compared with loading solution toassess the effect(s) of affinity chromatography step showed no change inthe kinetics of MT5 from different samples. The SPR sensorgrams of theVEGF MiniTrap constructs are shown in FIG. 38.

TABLE 7-6 VEGF MiniTrap k_(a) k_(d) K_(D) t_(1/2) Chi² R_(max) samples(10⁶ M⁻¹s⁻¹) (10⁻⁵ s⁻¹) (10⁻¹² M) (min) (RU²) (RU) Loading  9.44 1.741.84 664 0.10 20 solution column 2  8.83 1.49 1.69 775 0.17 28 eluatecolumn 1 12.18 1.80 1.48 641 0.18 19 eluate

7.9 Multiple Production Cycles

Chromatographic production of harvest as obtained by step 7.1 wascarried out using column 1 (hVEGF₁₆₅) and column 2 (mAb1) as shown in7.3. The columns were used for multiple chromatographic cycles. Theyields in the columns did not vary significantly due to additional runs,suggesting that the columns retained binding capacity (Table 7-7).

TABLE 7-7 Production Yield Affinity Column Column 1 Column 2 Run # 1 2 34 1 2 3 4 Load (mg) 21.2 19.7 19.7 19.7 21.2 19.7 19.7 19.7 Wash (mg)14.9 13.5 15.0 15.0 13.2 16.0 16.4 16.4 Eluate (mg) 4.8 5.4 5.2 4.8 1.61.8 1.8 1.8

HCP calculations in the loading solutions, wash fractions and elutedfractions for columns 1 and 2 were obtained using the method describedin 7.4 (FIG. 39). The total HCPs calculated showed that repeated use ofthe columns did not reduce the ability of the columns to bind toMiniTrap.

7.10 Optimizing the Affinity Chromatographic Columns

The chromatographic production of harvest material as obtained inSection 7.1 was performed using column 1 (VEGF₁₆₅) and column 2 (mAb1).For the optimization studies, 14 mg or 45 mg instead of 10 mg of theVEGF165 or the anti-VEGFR1 mAb were loaded onto two HiTrap NHS-ActivatedHP affinity columns (1 mL, GE Healthcare) and the columns were closed toallow coupling to take place for 30 minutes at room temperature. Thecolumn preparation and production of the harvest including the MiniTrapwas carried out as discussed in 7.2 and 7.3 above. The amount of MT5 inthe wash and eluted fractions is shown in Table 7-8. The comparison ofaffinity column with 14 mg or 45 mg (VEGF₁₆₅ or anti-VEGFR1 mAb (mAb1))conjugation amount instead of 10 mg shows an increased yield of MiniTrapfrom both columns. Thus, the column yield using the outlined method canbe improved by optimizing the protein to column ratio or by increasingthe conjugation efficiency by changing the pH, incubation time,incubation temperature, etc.

TABLE 7-8 Affinity Column (hVEGF₁₆₅) (mouse anti-VEGFR1 mAb) MW (kDa)~40 (Dimer) 145 Conjugation 10 14 10 45 Amount (mg) Load (mg) 21.2 45.521.2 45.5 Wash (mg) 14.9 36.8 13.2 29.2 Eluate (mg) ~5.0 7.6 ~1.8 5.57.11 Use of CEX with the Affinity Chromatography

A cell culture sample from MT5 expression was produced using column 1 asdiscussed in Section 7.3 above. The eluate obtained was subjected tocation exchange chromatography (CEX) column (HiTrap Capto S, 1 mL). Theoperating conditions of the column are shown in Table 7-9.

TABLE 7-9 Steps Affinity Cation Exchange (CEX) Column Affinity Column, 1mL HiTrap Capto S, 1 mL Load MT5 CM2926 20 mM Acetate, pH 5.0(Load/Wash1) Wash 1X DPBS pH 7.2 10 mM Phosphate, pH 7.0 ElutionPierce ™ IgG Elution 50 mM Tris, 62.5 mM Buffer (NH₄)₂SO₄, pH 8.5Regeneration/ 10 mM Glycine pH 2.5 50 mM Tris, 1M (NH₄)₂SO₄, Strip pH8.5

The total HCP in the original/starting cell culture sample, the affinitychromatography column 1 eluate and CEX eluate was about 230,000 ng/mL,about 9,000 ng/mL and about 850 ng/mL, respectively. The HCP amountswere quantitated determined using the Cygnus CHO HCP ELISA Kit, 3G, asmentioned above.

7.12 Use of Affinity Chromatography to Produce Other Anti-VEGF Proteins

Column 1 was evaluated to study its ability to produce other anti-VEGFproteins. Aflibercept and a scFv fragment with VEGF binding potentialwere used for this study. The production processes were carried out asdiscussed in Section 7.3. Table 7-10 demonstrates that column 1 wassuccessful in binding and eluting other anti-VEGF proteins.

TABLE 7-10 Affinity Column 1 scFv Aflibercept Load (mg) 10 20 Wash (mg)4.5 10.6 Eluate (mg) 3.6 10.2

Example 8 Mass Spectrometry-Based Characterization of VEGF MiniTrapConstructs

Materials. VEGF MiniTrap (MT1) was produced from aflibercept asdescribed in Example 1. VEGF MiniTrap 5 (MT5) was produced as describedin Example 7. VEGF MiniTrap (MT6) was produced by the following method:the coding regions of recombinant VEGF MiniTrap (MT5) were operablylinked to a signal sequence and cloned into a mammalian expressionvector, transfected into Chinese hamster ovary (CHO-K1) cells and stablytransfected pools were isolated after selection with 400 μg/mLhygromycin for 12 days. The stable CHO cell pools, grown in CDM, wereused to produce proteins for analysis.

8.1 Deglycosylation of Glycoproteins.

Samples from clarified harvest of MT1, MT5 and MT6 were diluted orreconstituted to a concentration of 0.52 mg/mL into a 28.8 μL solutionof 1% (w/v) RG surfactant (RapiGest SF, Waters, Milford, Mass.) and 50mM HEPES (pH 7.9). These solutions were heated to approximately 95° C.over 2 min, allowed to cool to 50° C., and mixed with 1.2 μL of PNGase Fsolution (GlycoWorks Rapid PNGase F, Waters, Milford, Mass.).Deglycosylation was completed by incubating the samples at 50° C. for 5min.

8.2 HILIC-Fluorescence-ESI-MS (MS/MS) Analysis.

MT1 was analyzed via HILIC separation combined with fluorescence andmass spectrometric detection. MT5 and MT6 were analyzed using onlyHILIC. Chromatography was performed using a Waters 2D Acquity UPLCequipped with photodiode array and fluorescence (FLR) detectors andinterfaced with a Waters Synapt G2-S mass spectrometer (MS conditions).A hydrophilic interaction chromatography (HILIC) mode of separation wasused with a Waters UPLC Glycan BEH Amide column, 150×2.1 mm, 1.7 μm. Thecolumn temperature was set to 60° C. and the autosampler temperature wasset to 5° C. The injection volume was 50 μL. The photodiode array scanrange was 190-700 nm. The FLR was set to excitation 265 nm, emission 425nm for RapiFluor-labeled glycans and excitation 274 nm, and emission 303nm for tyrosine present in the glycopeptides. The initial flow rate was0.4 mL/min with mobile phase A comprising of 100 mM ammonium formate (pH4.4) and mobile phase B being acetonitrile.

8.3 MS Conditions

Liquid chromatography/mass spectrometry (LC/MS) experiments wereconducted using a Waters Synapt G2-S mass spectrometer. The scan rangewas mass-to-charge ratio 100-2400 for positive and negative ion modeanalyses. Scan time was 1 s, and glu-fibrinopeptide B was constantlyinfused (2 μL/min) as a calibrant (“lock mass”). The capillary voltagewas set to 2.5 kV, with a source temperature of 120° C. and desolvationtemperature of 500° C. The nitrogen nebulizer gas flow was set to 700l/h.

8.4 Native SEC-MS

ACQUITY UPLC I class system (Waters, Milford, Mass.) was coupled to QExactive HF hybrid quadrupole-Orbitrap mass spectrometer (ThermoScientific, Bremen, Germany) for all online SEC-MS analyses. ACQUITYUPLC Protein BEH SEC Column (200 Å, 1.7 μm, 4.6×300 mm) was set at 30°C. and used for protein separation. The mobile phase was 100 mM ammoniumacetate at pH 6.8. Each separation was 30 minutes with a flow rate of0.3 mL/min, and the injection amount was set to 40 μg. The following MSparameters were used for online SEC-nano-ESI-MS data acquisition. Eachacquisition was 25 minutes beginning immediately after sample injection.The deglycosylated samples were ionized in positive mode with 3 kV sprayvoltage, 200° C. capillary temperature, and 70 S-lens RF level.In-source CID was set at 75 eV. Full MS scans were acquired at 15 Kresolving power with mass range between m/z 2000-8000. A maximuminjection time of 100 ms, automatic gain control target value of 3e6,and 10 microscans were used for full MS scans.

8.5 Peptide Mapping

Sample preparation for peptide mapping. Reduction was achieved by theaddition of 500 mmol/L dithiothreitol (DTT) to a final concentration of5 mmol/L followed by incubation at 4° C. for 60 min. Alkylation wasperformed by adding 500 mmol/L iodoacetamide (IAM) to a finalconcentration of 10 mmol/L and incubating at 4° C. for 60 min in thedark. The denaturing buffer was exchanged for digestion buffer (1 mol/Lurea in 0.1 mol/L Tris, pH 7.8) using Zeba™ Spin 7 K MWCO size-exclusiondesalting columns (P/N 89882) (Thermo Scientific, Waltham, Mass.)according to the manufacturer's instructions. Recombinant porcinetrypsin (purchased from Sigma, Cat #03708985001) was added at a 1:18(enzyme: sample) mass ratio (based on VEGF MiniTrap proteinconcentration as measured by UV-Vis spectrophotometry after bufferexchange), the concentration of VEGF MiniTrap proteins was adjusted to0.5 μg/μL and digestion allowed to proceed during a 4 h incubation atroom temperature. When the digestion was complete, 0.1% formic acid inLC-MS grade water was added at a 1:1 volume ratio. Digests were storedat −80° C. until analysis.

LC-MS/MS analysis of tryptic digests. One or more 2.5 μg (10 μL) ofpeptide digests were loaded via autosampler onto a C18 column enclosedin a thermostatted column oven set to 40° C. Samples were held at 7° C.while queued for injection. The chromatographic method was initiatedwith 98% Mobile Phase A (0.1% volume fraction of formic acid in water)and 2% Mobile Phase B (0.1% volume fraction of formic acid inacetonitrile) with the flow rate set at a constant 0.200 mL/min. After a10 min wash, peptides were eluted over a 110 min gradient in whichMobile Phase B content rose at a rate of 0.39% per min to reach a finalcomposition comprising 45% Mobile Phase B. Prior to the next sampleinjection, the column was washed for 15 min with 97% Mobile Phase B,then equilibrated at 98% Mobile Phase A for 25 min. The eluate wasdiverted to waste for the first 1.5 minutes and final 5 minutes of therun. Peptides eluting from the chromatography column were analyzed by UVabsorption at 214 nm followed by mass spectrometry on the LTQ OrbitrapElite or Discovery XL. Replicate peptide mapping data were collected forPS 8670 and RM 8671 samples to include three tandem MS (MS/MS) analysesand one MS-only analysis each. The MS/MS analyses were performed forpeptide identification in data-dependent mode in which one cycle ofexperiments consisted of one full MS scan of 300 m/z to 2000 m/zfollowed by five sequential MS/MS events performed on the first throughfifth most intense ions detected at a minimum threshold count of 500 inthe MS scan initiating that cycle. The sequential mass spectrometry(MS^(n)) AGC target was set to 1E4 with microscans=3. The ion trap wasused in centroid mode at normal scan rate to analyze MS/MS fragments.Full MS scans were collected in profile mode using the high resolutionFTMS analyzer (R=30,000) with a full scan AGC target of 1E6 andmicroscans=1. Ions were selected for MS/MS using an isolation width of 2Da, then fragmented by collision induced dissociation (CID) with heliumgas using a normalized CID energy of 35, an activation Q of 0.25 and anactivation time of 10 msec. A default charge state was set at z=2. Datadependent masses were placed on the exclusion list for 45 s if theprecursor ion triggered an event twice within 30 s; the exclusion masswidth was set at ±1 Da. Charge state rejection was enabled forunassigned charge states. A rejection mass list included commoncontaminants at 122.08 m/z, 185.94 m/z, 355.00 m/z, 371.00 m/z, 391.00m/z, 413.30 m/z, 803.10 m/z, 1222.10 m/z, 1322.10 m/z, 1422.10 m/z,1522.10 m/z, 1622.10 m/z, 1722.10 m/z, 1822.10 m/z, and 1922.10 m/z.MS-only analyses were performed for the generation of the TICnon-reduced peptide map and reduced maps.

8.6 Results

Structure of VEGF MiniTrap constructs. Structure of VEGF MiniTraps MT1,MT5 and MT6 are depicted in FIG. 40, FIG. 41, FIG. 43 and FIG. 44.

Initial mass analysis using SEC-MS confirmed the identities of all threemolecules at intact protein level after deglycosylation (FIG. 42). TheTotal Ion Chromatogram (TIC) of the native SEC-MS analysis demonstratesdetection of an intact VEGF MiniTrap molecules at around 12-13 minutes.The expansion of the low molecular weight (LMW) region of the TIC showedpresence of LMW impurities in all the three protein samples.

The deconvoluted mass spectra for the VEGF MiniTraps further confirmedtheir identity and provided data for elucidation of the major PTMspresent in the samples comprising MT1 and MT5 (FIG. 43), which aredimers and MT6 (FIG. 44) which is a single chain protein.

Analysis of MT1 sample. The LMW species identified from the TIC of theSEC-MS analysis of the samples comprising MT1 was extracted to examinethree distinct LMW impurities—LMW1, LMW2, and LMW3 (FIG. 45A and FIG.45B). LMW1 species comprised a truncated species of aflibercept. LMW2species comprised the Fc impurity present in the sample form thecleavage of aflibercept which was performed to produce MiniTrap. LMW3species comprised a monomer possibly cleaved from the MT1 (dimer)molecule.

MT1 sample did not show presence of FabRICATOR enzyme, which had beenused to cleave aflibercept to form a MiniTrap protein. The enzyme, ifpresent, is detected at about 11.5 and 12.5 minutes. No such peak wasdetected during the SEC-MS analysis of the MT1 sample (FIG. 46).

Analysis of MT5 sample. The LMW species identified from the TIC of theSEC-MS analysis of the samples comprising MT5 was extracted to examinethe presence of two distinct LMW impurities—LMW1 and LMW2 (FIG. 47).

Analysis of MT6 sample. The LMW species identified from the TIC of theSEC-MS analysis of the samples comprising MT1 was extracted to examinethe presence of three distinct LMW impurities—LMW1, LMW2, and LMW3 (FIG.48). LMW2 species comprised a fragment of the MT6 wherein the cleavageproduced the fragment of VEGF MiniTrap with the G45 linker (SEQ ID NO.:111). LMW5 species comprised a fragment of the MT6 wherein the cleavageoccurred right before or after the G45 linker (SEQ ID NO.: 111).

The glycans in the MT6 sample were identified by their mass and elutionorder in the HILIC chromatography method using the glucose unit valuepioneered by Waters and the National Institute for BioprocessingResearch and Training (Dublin, Ireland) (FIG. 49A and FIG. 49B).

Free thiol Quantification. Cysteine residues of the VEGF MiniTrapconstructs may be involved in the formation of intra- andinter-molecular disulfide bond(s) or they may exist as free thiols. Thepresence of sulfide bonds in peptides and proteins has been shown toimpose conformational rigidity on a protein. Thiols can be detected by avariety of reagents and separation techniques. The analysis of the threeVEGF MiniTrap constructs for a very low level of free thiols is shown inTable 8-1.

TABLE 8-1 Peptide Location (site of free cysteine) MT1 MT5 MT6 VEGFR1ELVIPCR <0.1% <0.1% <0.1% (SEQ ID NO.: 81) VEGFR2 LVLNCTAR  0.3%  0.3% 0.3% (SEQ ID NO.: 82) Fc Hinge THTCPPCPAPELLG  0.0%  0.0% N/A(SEQ ID NO.: 83)

Trisulfide Quantification. Similar to free thiols in Cys residues of theVEGF MiniTrap constructs, trisulfide bonds can influence the structureof the protein. The analysis of the three VEGF MiniTrap constructs underconditions with very low level of free thiols is shown in Table 8-2.

TABLE 8-2 Location Peptide MT1 MT5 MT6 VEGFR1 ELVIPCR-EIGLLTCEATVNGHLYK 0.1% <0.1%  0.1% (SEQ ID NO.: 84) VEGFR2 LVLNCTAR-SDQGLYTCAASSGLMT<0.1% <0.1% <0.1% K(K) (SEQ ID NO.: 85) Fc HingeTHTCPPCPAPELLG-THTCPPCPAP 1.5% 3.7% N/A ELL(G) (SEQ ID NO.: 86)

Intra-chain disulfide in the Hinge region. Mispaired disulfide bonds inthe hinge region can have implications on the structure, function andstability of the VEGF MiniTrap constructs. The analysis of the threeVEGF MiniTrap constructs for a very low or no intra-chain disulfidebinds in the hinge region of the VEGF MiniTrap constructs[THTC*PPC*PAPELLG, C* shows where intra-chain sulfide bond can beformed] (SEQ ID NO.: 83) is shown in Table 8-3.

TABLE 8-3 Peptide MT1 MT5 MT6 Disulfide <0.1% <0.1% N/A Trisulfide <0.1%<0.1% N/A

Cross and parallel disulfide linkage isomer quantification. For MT1 andMT5, which are dimers connected by parallel disulfide bonds in the hingeregions, there is a possibility of isomers wherein the disulfide bondsin the hinge region can be crossed (FIG. 50).

The quantification of types of disulfide bond, parallel versus cross,showed that MT5 recombinantly expressed protein had a slightly higherlevel of cross disulfide bridge in the Fc hinge region compared to theMT1—which is a FabRICATOR digested molecule (Table 8-4).

TABLE 8-4 Disulfide MT1 MT5 MT6 Cross 0.2% 3.9% N/A Parallel 99.8% 96.1%N/A

Post-Translational Modifications (PTMs).

TABLE 8-5 PTM Site Modified Peptide MT1 MT5 MT6 Deamidation Asn84EIGLLTCEATV

GHLYK Succinimide  3.1%  3.2%  3.2% (Asn319) (SEQ ID NO.: 87)Asp/iso Asp 21.9% 18.9% 20.9% Asn99 QT

TIIDVVLSPSHGIELSVGEK Succinimide  4.6%  4.6%  4.0% (Asn334)(SEQ ID NO.: 88) Asp/iso Asp  0.7%  0.5%  0.6% Oxidation Met10SDTGRPFVE 

YSEIPEIIHMTEGR  1.8%  2.1%  2.1% (SEQ ID NO.: 89) Met20SDTGRPFVEMYSEIPEIIH 

TEGR  2.9%  3.0%  2.7% (SEQ ID NO.: 90) Met245GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSS — —  1.4% DTGRPFVE 

YSEIPEIIHMTEGR (SEQ ID NO.: 91) Met255 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSS ——  2.7% DTGRPFVE 

YSEIPEIIHMTEGR (SEQ ID NO.: 92) Met163 TQSGSE 

K  4.3%  4.3%  3.8% (Met398) (SEQ ID NO.: 93) Met192 SDQGLYTCAASSGL

TK  5.0%  5.0%  4.2% (Met427) (SEQ ID NO.: 94) C-term Gly211THTCPPCPAPELL

 0.1%  2.0% — Glycine loss (SEQ ID NO.: 95)

Evaluation of PTMs in all the three VEGF MiniTrap constructs showedcomparable levels of PTMs (Table 8-5). The deamidation observed at Asn84to form succinimide was in the range of about 3.1-3.2% and to formaspartic acid/iso aspartic acid was 18.9-21.9%. Oxidation of severalmethionine residues (e.g., Met10, Met 20m Met163 and Met192) wasobserved in the range of about 0.7-6.8% for all the three VEGF MiniTrapconstructs. MT6, which, in contrast to MT1 and MT5, comprises a linker,showed additional oxidation of methionine residues on the linker (e.g.,Met245 and Met255). About 0.1% and 2.0% of the C-terminal glycine(Gly211) in MT1 and MT5 showed a glycine loss. This was not observed forMT6, which lacks a C-terminal glycine.

Advanced glycation end-product modifications related to lysine andarginine glycation. Glycation of the VEGF MiniTrap constructs can altertheir structure and function, leading to impaired anti-VEGF activity.

TABLE 8-6 Site PTM MT1 MT5 MT6 Arg5 3-Deoxyglucosone   8.0%   8.1%  9.2% Glycation   0.1%   0.1%   0.1% Carboxymethylation   1.5%   1.4%  1.4% Arg153 3-Deoxyglucosone <0.1% <0.1% <0.1% Arg96 3-Deoxyglucosone<0.1% <0.1% <0.1% Lys62 Glycation   1.1%   1.1%   1.3%Carboxymethylation <0.1% <0.1% <0.1% Lys68 Glycation   0.4%   0.3%  0.5% Lys149 Glycation   0.6%   0.5%   0.6% Carboxymethylation <0.1%<0.1% <0.1% Lys185 Glycation <0.1% <0.1% <0.1%

Evaluation of modifications in all three VEGF MiniTrap constructs showedcomparable levels (Table 8-6).

Modified sites. The modified sites on the VEGF MiniTrap constructs, aselucidated by the intact mass analysis as per Section 8.4, wereconfirmed and quantified using reduced peptide mapping as illustrated inSection 8.5 (Table 8-7). The site T90N91 for peptide sequence TNYLTHR(SEQ ID NO.: 21), the ** represents that asparagine was converted toaspartic acid after truncation, whereas for site N99T100 the peptidesequence QTNTIIDVVLSPSHGIELSVGEK (SEQ ID NO.: 19), the * represents ahigh level of no-specific cleavage by trypsin. These two truncationsites were found to form LMW species impurities during evaluation of MT1and MT5. The truncation at M245Y246 was found only on MT6 which had theunique linker and was responsible for the LMW2 species impurity duringthe MT6 preparation.

TABLE 8-7 Site Peptide Sequence MT1 MT5 MT6 N99T100QTNTIIDVVLSPSHGIELSVGEK* 12.6% 13.2% 13.6% (SEQ ID NO.: 96) T90N91TNYLTHR** (SEQ ID  0.5%  0.1%  0.3% NO.: 97) M245Y246GGGGSGGGGSGGGGSGGGGSGGGGS — —  1.8% GGGGSSDTGRPFVEMYSEIPEIIHM TEGR(SEQ ID NO.: 98) M10Y11 SDTGRPFVEMYSEIPEIIHMTEGR  0.2%  1.5%  1.7%(SEQ ID NO.: 99)

-   -   Glycosites occupancy quantification. N-glycosylation is a common        PTM. Characterizing the site-specific N-glycosylation including        N-glycan macroheterogeneity (glycosylation site occupancy) and        microheterogeneity (site-specific glycan structure) is important        for the understanding of glycoprotein biosynthesis and function.        The extent of glycosylation can change depending on how the        protein is expressed. The levels of glycosylation at N36 were        similar for all the three VEGF MiniTraps (Table 8-8 and FIG.        51). Similarly, the levels of glycosylation at N68 were also        similar for all the three VEGF MiniTraps (Table 8-8 and FIG.        52). The levels of glycosylation at N123 were also similar for        all the three VEGF MiniTraps (Table 8-8 and FIG. 53), but        mannose-5 was found to be elevated in the MT1 preparation. For        the VEGF MiniTrap constructs, glycosylation at Asn196 was lower        for MT5 and MT6, compared to MT1 (Table 8-8 and FIG. 54).        Additionally, the mannose-5 was also elevated for the MT1        preparation than MT5 and MT6 preparations.

TABLE 8-8 Site Peptide MT1 MT5 MT6 N36 (R)VTSPNITVTLK 98.3% 98.1% 99.4%(SEQ ID NO.: 100) N68 (K)GFIISNATYK 51.9% 55.4% 64.9% (SEQ ID NO.: 101)N123 (K)LVLNCTAR 99.9% 99.4% 98.4% (SEQ ID NO.: 102) N196 (K)NSTFVR98.6% 44.5% 55.1% (SEQ ID NO.: 103)

-   -   Analysis of N-glycans. The glycosylation at N36 is shown in        Table 8-9. G2F, G2FS, G2FS2 were the major N-glycans found in        all the three VEGF MiniTraps. For glycosylation at N68 shown in        Table 8-10, G2F and G2FS were the major N-glycans found in all        the three VEGF MiniTraps. For glycosylation at N123 is shown in        Table 8-11, G2F and G2S were the major N-glycans found in all        the three VEGF MiniTraps and Mannose-5 was detected at high        levels in MT1 compared to MT5 and MT6. For glycosylation at N196        shown in Table 8-12, G2, G2S, G2S2 were the major N-glycans        found in all the three VEGF MiniTraps and Mannose-5 was detected        at high levels in MT1 compared to MT5 and MT6.

TABLE 8-9 Glycans at N36 MT1 MT5 MT6 G0F-GlcNAc  2.0%  1.8%  1.8% G1F 3.2%  1.0%  1.4% G1F-GlcNAc  4.8%  4.6%  4.9% G1FS-GlcNAc  3.1%  3.8% 3.1% G2F 17.4% 15.1% 19.8% G2F2S  1.7%  2.0%  2.2% G2FS 34.2% 31.5%31.9% G2FS2 20.4% 25.8% 19.0% G3FS  2.3%  4.0%  5.5% G3FS2  2.6%  4.7% 5.0% G3FS3  1.1%  2.4%  1.9% G1_Man5 + Phos  1.2%  0.3%  0.2% Man6 +Phos  5.7%  2.5%  2.8%

TABLE 8-10 Glycans at N68 MT1 MT5 MT6 G0F-GlcNAc  1.2%  1.1%  1.1% G1F 5.1%  1.4%  1.7% G1F-GlcNAc  3.9%  3.9%  4.0% G1FS  1.2%  0.4%  0.4%G1FS1-GlcNAc  1.2%  1.6%  1.4% G2F 27.4% 23.6% 28.6% G2F2S  2.2%  3.0% 3.4% G2FS 52.4% 55.2% 50.2% G2FS2  3.9%  6.9%  5.8% G3FS  0.5%  1.2% 1.6% G3FS2  0.4%  1.1%  1.2%

TABLE 8-11 Glycans at N123 MT1 MT5 MT6 G0-GlcNAc  3.5%  3.7%  3.5%G1-GlcNAc  6.2%  6.8%  6.4% G1S-GlcNAc  4.1%  3.5%  2.8% G2 10.6% 16.7%17.1% G2F  1.5%  7.2%  7.0% G2FS  2.1% 13.6% 14.2% G2S 12.7% 26.1% 25.5%G2S2  1.3%  5.0%  6.6% G1_Man4  3.8%  1.3%  1.4% G1S_Man4  3.9%  2.1% 1.8% G1_Man5  4.0%  1.2%  1.1% G1S_Man5  3.2%  1.4%  1.4% Man4  2.6% 1.9%  1.8% Man5 35.5%  4.3%  3.1% Man6  1.1%  0.1%  0.1% Man7  2.8% 0.1%  0.1%

TABLE 8-12 Glycans at N196 MT1 MT5 MT6 G0-GlcNAc  1.9%  1.8%  1.9% G1 4.1%  3.6%  4.2% G1-GlcNAc  1.9%  2.5%  2.4% G1S-GlcNAc  2.9%  2.6% 1.8% G2 20.7% 28.2% 32.1% G2F  2.0%  5.1%  6.0% G2FS  2.0%  6.1%  6.2%G2FS2  0.5%  1.6%  1.3% G2S 17.7% 31.2% 29.9% G2S2  4.4%  9.7%  6.7% G3S 0.1%  0.7%  1.0% G1S_Man4  1.0%  0.3%  0.3% G1_Man5  2.3%  0.5%  0.5%Man3  3.1%  0.7%  0.6% Man4  2.7%  0.8%  0.6% Man5 30.4%  3.6%  3.4%

O-glycans at the linker for MT6. The GS linker for MT6 was evaluated tostudy O-glycans on MT6. O-xylosylation was found to on serine residueslocated on the GS linker of MT6(GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGR, underlinedserine residues were glycosylated) (SEQ ID NO.: 98). The composition ofthe O-glycans is shown in Table 8-13.

TABLE 8-13 Composition Mass Annotation Number Level Xylosylation +132.0

Tri <0.1% di   1.5% mono    15% Xylose + Galactose +294.1

mono   0.9% Xylose + Galactose + +585.2

mono   0.7% Sialic Acid

HILIC-FLR-MS analysis. HILIC-FLR-MS analysis was performed for all theVEGF MiniTrap proteins as described in Section 8.2. The analysis showedthat the N-linked glycans for MT5 and MT6 were similar but weredifferent than the ones obtained for MT1 (FIG. 55 shows the full scaleand stacked chromatograms, FIG. 56 shows full scale and overlaidchromatograms and FIG. 57 shows the full scale, stacked and normalizedchromatograms).

Finally, the percent glycosylation and detailed glycan identificationand quantification for all three VEGF MiniTrap proteins is listed inTable 8-14 and FIG. 58A-C, respectively. As observed in all the glycananalysis, the glycosylation profile and mannose levels for MT5 and MT6are similar, but different from MT1.

TABLE 8-14 MT1 MT5 MT6 % Fucosylation 42.9% 57.8% 57.2% %Galactosylation 71.6% 92.9% 93.7% % Sialylation 33.1% 47.6% 44.8% % HighMannose 17.6%  2.6%  2.3% % Bisecting  1.9%  0.4%  0.4%

Example 9 Production and Color Quantification Using Upstream Medium andFeed Process Optimization (A) Un-Optimized CDM (Control Bioreactor)

The manufacture of MiniTrap described in Example 5 was employed.

The operating parameters for the study steps are as known to one ofordinary skill in the art.

Medium at day 0=CDM1 and included the following nutrients, antioxidantsand metals:

-   -   Cysteine was added at a cumulative concentration of 8-9 mM    -   Metals in Starting Medium are listed below at lx concentration        (where the concentrations are prior to inoculum addition):        -   Fe=68-83 micromoles per liter of culture        -   Zn=6-7 micromoles per liter of culture        -   Cu=0.1-0.2 micromoles per liter of culture        -   Ni=0.5-1 micromoles per liter of culture

On harvesting MT1, the production procedure as shown in FIG. 59 wasfollowed. The operating parameters for the chromatography are known toone of ordinary skill in the art. The operating parameters for theaffinity capture (step 3 of FIG. 59), affinity Flowthrough (step 5 ofFIG. 59), AEX (step 8 of FIG. 59), and HIC (step 9 of FIG. 59) areoutlined in Table 9-1. The proteolytic cleavage of aflibercept followingaffinity capture and filtration step was carried out using the procedureas outlined in Example 1.2.

TABLE 9-1 Affinity Affinity Steps Capture flowthrough AEX HIC ResinMabSelect SuRe MabSelect SuRe POROS 50 HQ Capto Phenyl HS Load  30 g/Lresin  30 g/L resin 40 g/L resin 100 g/L resin pH 6.80-7.20 pH8.30-8.50, pH 4.40-4.60 1.90-2.10 7.50-10.50 mS/cm mS/cm Equilibration 20 mM Sodium  26 mM Tris, 16 50 mM Tris  40 mM Tris, 30 Phosphate pH mMSodium pH 8.30-8.50, mM Sodium 7.10-7.30, Phosphate, 18 1.90-2.10Citrate, 74 2.60-3.20 mM Acetate pH mS/cm mM Acetate pH mS/cm 6.90-7.10,4.40-4.60, 2.00-4.00 7.50-10.50 mS/cm mS/cm Wash 1  10 mM Sodium  26 mMTris, 16 50 mM Tris  40 mM Tris, 30 Phosphate, 500 mM Sodium pH8.30-8.50, mM Sodium mM NaCl pH Phosphate, 18 1.90-2.10 Citrate, 747.10-7.30, mM Acetate pH mS/cm mM Acetate pH 40-50 6.90-7.10, 4.40-4.60,mS/cm 2.00-4.00 7.50-10.50 mS/cm mS/cm Wash 2  20 mM Sodium N/A N/A N/APhosphate pH 7.10-7.30, 2.60-3.20 mS/cm Elution  40 mM Acetic  40 mMAcetic N/A N/A Acid pH Acid pH 2.80-3.20, 2.80-3.20, 0.28-0.36 0.28-0.36mS/cm mS/cm Regeneration/ 500 mM Acetic 500 mM Acetic  2M SodiumProprietary Strip 1 Acid, pH Acid, pH Chloride (NaCl) buffer 2.25-2.65,2.25-2.65, 0.90-1.25 0.90-1.25 mS/cm mS/cm Regeneration/ N/A N/A  1NSodium N/A Strip 2 Hydroxide (NaOH)

Table 9-2 shows the color quantification of the pools obtained onperforming various chromatographic steps. The color quantification wascarried using samples from the pool having a protein concentration of 5g/L.

Affinity Capture Pool refers to the eluate collected on performing theaffinity capture step (step 3 of FIG. 59). Enzymatic Pool refers to theflowthrough collected on performing the enzymatic cleavage step (step 4of FIG. 59). Affinity flowthrough Pool refers to the flowthroughcollected on performing the affinity flowthrough step (step 5 of FIG.59) and Affinity flowthrough Eluate refers to the eluate collected onperforming the affinity flowthrough step (step 5 of FIG. 59). AEX Pooland AEX Strip refer to the flowthrough and stripped fractions obtainedon performing anion exchange chromatography step (step 8 of FIG. 59).HIC Pool refers to the flowthrough collected on performing thehydrophobic interaction chromatography step (step 9 of FIG. 59).

Each step as seen in Table 9-2 shows a reduction in coloration (asobserved from the reduction in b* values of the pools). For example, onperforming affinity flowthrough chromatography, the flowthrough fractionhas a b* value of 2.16 (reduced from a b* value of 2.52 for theflowthrough collected from the affinity capture step). The flowthroughand wash following the AEX separation further reduced the coloration, asobserved by reduction in the b* value from 2.16 to 0.74. As expected,stripping the AEX column led to a sample with a yellow-brown color whichwas significantly more intense than the coloration from the flowthroughand wash following the AEX separation as seen from the b* values (8.10versus 0.74). Lastly, a HIC step afforded a further reduction in color(the b* value can be normalized for 5 g/L protein concentration from theb* value obtained for HIC pool at 28.5 g/L protein concentration).

TABLE 9-2 Color Quantification of Samples at Various Production StepsSample Conc. (g/L) L* a* b* Affinity Capture Pool 5.0 ± 0.1 98.75 −0.122.52 Enzymatic Cleavage Pool 5.0 ± 0.1 99.03 −0.07 1.61 Affinityflowthrough Pool 5.0 ± 0.1 98.95 −0.08 2.16 Affinity flowthrough Eluate5.0 ± 0.1 98.92 −0.01 0.83 AEX Pool 5.0 ± 0.1 99.72 −0.03 0.74 AEX 2MNaCl Strip 5.0 ± 0.1 96.25 −0.42 8.10 HIC Pool 28.5 98.78 −0.28 3.11

(B) Optimized CDM (Low Cysteine, Low Metals and Increased AntioxidantsBioreactor)

The effect of lowering the concentration of cysteine, lowering theconcentration of metals, and increasing antioxidants on coloration wasevaluated using the following protocols:

Medium at day 0=CDM1

-   -   Cysteine was added at a cumulative concentration of 5-6 mM    -   Antioxidants were added to CDM1 to reach the following        cumulative concentrations (where the concentrations are prior to        inoculum addition):        -   Taurine=10 mM of culture        -   Glycine=10 mM of culture        -   Thioctic Acid=0.0024 mM of culture        -   Vitamin C (ascorbic acid)=0.028 mM of culture    -   Metals in Starting Medium are listed below for the 1× level.        -   Fe=68-83 micromoles per liter of culture        -   Zn=6-7 micromoles per liter of culture        -   Cu=0.1-0.2 micromoles per liter of culture        -   Ni=0.5-1 micromoles per liter of culture.        -   The reduction of all the metals included using 0.25× the            concentrations noted above for the medium.

Upon harvesting of the MT1 sample, the production procedure as shown inFIG. 59 was followed. The operating parameters for the chromatographyare known to one of ordinary skill in the art. The operating parametersfor the affinity capture, affinity flowthrough, and HIC are outlined inTable 9-1. The proteolytic cleavage of aflibercept following affinitycapture and filtration step was carried out using the procedure asoutlined in Example 1.2.

Table 9-3 shows the color quantification of the pools obtained onperforming the various chromatographic steps. The color quantificationwas carried using samples from the pool having a protein concentrationof 5 g/L. The steps as seen in Table 9-3 afforded a similar productionas seen for steps in Table 9-2.

TABLE 9-3 Color Quantification of Samples at Various Production Steps ofMiniTrap Sample Conc. (g/L) L* a* b* Affinity Capture Pool 5.0 ± 0.199.18 −0.09 1.77 Enzymatic Cleavage Pool 5.0 ± 0.1 99.44 −0.06 1.17Affinity flowthrough Pool 5.0 ± 0.1 99.32 −0.10 1.58 Affinityflowthrough Eluate 5.0 ± 0.1 99.74 −0.05 0.60 AEX Pool 5.0 ± 0.1 99.63−0.07 0.50 AEX 2M NaCl Strip 5.0 ± 0.1 97.63 −0.49 6.10 HIC Pool 27.699.07 −0.29 2.32

Comparing Table 9-2 and Table 9-3, it is evident that the “Low Cysteine,Low Metals, and Increased Antioxidants Bioreactor Condition” had lowercolor in affinity capture pool (b* value of 1.77) compared to the“Control Bioreactor Condition” (b* value 2.52).

An MT sample with a concentration of 160 g/L, where the MT is formedusing the steps listed in Table 9-2 and Table 9-3, is predicted to havea b* value of 13.45 for the “Low Cysteine, Low Metals, and IncreasedAntioxidants Bioreactor Condition” and a b* value of 17.45 for the“Control Bioreactor Condition.” A 23% reduction in color is achievedthrough optimization of the upstream media and feeds. Similarly, an MTsample with a concentration of 110 g/L, where the MT is formed using thesteps listed in Table 9-2 and Table 9-3, is predicted to have a b* valueof 9.25 for the “Low Cysteine, Low Metals, and Increased AntioxidantsBioreactor Condition” and a b* value of 12 for the “Control BioreactorCondition.”

To understand how each production unit operation contributes to colorreduction, the b* value for each production process intermediate as apercentage of the color of affinity capture pool was calculated (Table9-4).

TABLE 9-4 b* as % of Affinity Conc. Capture Sample (g/L) b* Δb* PoolControl Affinity Capture 5.0 ± 0.1 2.52 N/A 100.0 Bioreactor PoolEnzymatic 5.0 ± 0.1 1.61 −0.91 63.8 Cleavage Pool Affinity 5.0 ± 0.12.16 0.55 85.7 flowthrough Pool AEX Pool 5.0 ± 0.1 0.74 −1.42 29.4 HICPool 5.0 ± 0.1 0.55 −0.19 21.8 Low Cysteine, Affinity Capture 5.0 ± 0.11.77 N/A 100.0 Low Metals, Pool and Increased Enzymatic 5.0 ± 0.1 1.17−0.60 66.1 Antioxidants Cleavage Pool Bioreactor Affinity 5.0 ± 0.1 1.580.41 89.2 flowthrough Pool AEX Pool 5.0 ± 0.1 0.50 −1.08 28.2 HIC Pool5.0 ± 0.1 0.42 −0.08 23.7

The AEX unit operation provides the most color reduction (1.08 to 1.42change in b*) while the HIC unit operation provides some additionalcolor reduction (0.08 to 0.19 change in b*). The unit operationsevaluated overall remove 76.3%-78.2% of the color present in affinitycapture pool.

The color of various production process intermediates for “ControlBioreactor Condition” and “Low Cysteine, Low Metals, and IncreasedAntioxidants Bioreactor Condition” were also studied for the percentageof 2-oxo-histidines and percentage of oxo-tryptophans in theoligopeptides that were generated by protease digestion, as measured bymass spectrometry as shown in Table 9-5 and Table 9-6, respectively. Thepeptide mapping was performed as discussed in Example 3.

Referring to Table 9-5, on comparing the histidine oxidation levels inthe pools in different production steps, it is evident that relativeabundance of the percentage of histidine oxidation levels for MT formedreduces in the pool as the production process progresses. For example,for H209 in the “Control Bioreactor Condition”, the percent histidineoxidation level was 0.062 for the enzymatic cleavage pool and this wasreduced to 0.029 for AEX flowthrough and further reduced to 0.020 forthe HIC pool. Similarly, for H209 in the “Low Cysteine, Low Metals, andIncreased Antioxidants Bioreactor Condition”, the percent histidineoxidation level was 0.039 for the enzymatic cleavage pool and this wasreduced to 0.023 for AEX flowthrough and further reduced to 0.016 forthe HIC pool. Thus, the production strategy led to a reduction inpercentage of histidine oxidation levels in MT. As the colorationreduced, presence of some of the oxidized residues in the sample alsoreduced. Similar to histidine oxidation, tryptophan oxidation levelswere also tracked for the pools in different production steps for boththe “Control Bioreactor Condition” and “Low Cysteine, Low Metals, andIncreased Antioxidants Bioreactor Condition” (Table 9-6).

TABLE 9-5 Histidine Oxidation Levels (%) H19 H86 H95 H110 H145 H209Fraction Color (b*) (+14) (+14) (+14) (+14) (+14) (+14) ControlEnzymatic 1.61 0.023 0.018 0.011 0.014 0.007 0.062 Bioreactor CleavagePool Condition Affinity 2.16 0.030 0.027 0.018 0.015 0.011 0.067flowthrough Pool (AEX Load) Affinity 0.83 0.030 0.022 0.000 0.018 0.0040.046 flowthrough Eluate AEX 0.74 0.026 0.025 0.013 0.016 0.010 0.029flowthrough AEX 2 M 8.10 0.024 0.063 0.033 0.019 0.012 0.063 NaCl StripHIC Pool 0.55 0.018 0.009 0.002 0.021 0.005 0.020 Low Enzymatic 1.170.019 0.017 0.009 0.014 0.008 0.039 Cysteine, Cleavage Pool 1.58 0.0260.025 0.013 0.014 0.010 0.043 Low Metals, Affinity and flowthroughIncreased Pool (AEX Antioxidants Load) Bioreactor Affinity 0.60 0.0310.017 0.007 0.020 0.003 0.016 Condition flowthrough Eluate AEX 0.500.020 0.022 0.009 0.014 0.010 0.023 flowthrough AEX 2 M 6.10 0.020 0.0550.025 0.016 0.011 0.042 NaCl Strip HIC Pool 0.42 0.013 0.009 0.002 0.0170.003 0.016

TABLE 9-6 Tryptophan Oxidation Levels (%) W58 W58 W58 W58 W138 W138 W138Fraction Color (b*) (+4) (+16) (+32) (+48) (+4) (+16) (+32) ControlEnzymatic 1.61 0.006 0.032 0.289 0.000 0.020 1.093 0.106 BioreactorCleavage Condition Pool Affinity 2.16 0.016 0.055 0.327 0.000 0.0170.771 0.111 flowthrough Pool (AEX Load) Affinity 0.83 0.009 0.031 0.4530.000 0.025 1.039 0.132 flowthrough Eluate AEX 0.74 0.014 0.038 0.2830.000 0.023 0.720 0.120 flowthrough AEX 2 M 8.10 0.043 0.089 0.462 0.0000.031 0.620 0.175 NaCl Strip HIC Pool 0.55 0.037 0.126 0.413 0.000 0.0200.656 0.274 Low Enzymatic 1.17 0.009 0.027 0.239 0.001 0.027 1.026 0.136Cysteine, Cleavage Low Metals, Pool and Affinity 1.58 0.013 0.045 0.2840.000 0.021 0.628 0.107 Increased flowthrough Antioxidants Pool (AEXBioreactor Load) Condition Affinity 0.60 0.003 0.026 0.421 0.021 0.0251.032 0.132 flowthrough Eluate AEX 0.50 0.011 0.031 0.235 0.000 0.0220.676 0.102 flowthrough AEX 2 M 6.10 0.034 0.073 0.478 0.000 0.032 0.6350.169 NaCl Strip HIC Pool 0.42 0.029 0.122 0.355 0.000 0.022 0.800 0.236

What is claimed is:
 1. A method of producing aflibercept, comprising: a)producing a clarified harvest of cells cultured in a chemically definedmedium (CDM); b) binding aflibercept from said clarified harvest to anaffinity chromatography column comprising a polypeptide capable ofbinding to or interacting with said aflibercept, wherein saidpolypeptide is an antibody, a fusion protein, a ScFv or a fragmentthereof; c) eluting said aflibercept of step (b) forming an affinityeluate, wherein said eluate has a first color; d) subjecting said eluatecomprising aflibercept to anion exchange chromatography (AEX); and e)collecting a flowthrough fraction, wherein said flowthrough fraction hasa second color, wherein the first color is more yellow brown than thesecond color.
 2. The method of claim 1, wherein said cell is selectedfrom a group consisting of CHO, NS0, Sp2/0, embryonic kidney cells andBHK.
 3. The method of claim 1, wherein said clarified harvest comprisesone or more aflibercept variants, wherein said variants have at leastone oxidized amino acid residue.
 4. The method of claim 3, wherein saidoxidized amino acid residue is selected from group consisting ofmethionine, tryptophan, histidine, phenylalanine, tyrosine and acombination thereof.
 5. The method of claim 3, wherein said oxidizedamino acid residue is histidine.
 6. The method of claim 3, wherein saidoxidized amino acid residue is tryptophan.
 7. The method of claim 3,wherein said aflibercept variant is selected from an amino acid residueon a polypeptide having an amino acid sequence as set forth in the groupconsisting of: SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ IDNO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, SEQ ID NO.: 56,SEQ ID NO.: 62, SEQ ID NO.: 63, SEQ ID NO.: 64, SEQ ID NO.: 65, SEQ IDNO.: 66, SEQ ID NO.: 67, SEQ ID NO.: 68, SEQ ID NO.: 69, SEQ ID NO.: 70,SEQ ID NO.: 71 and combinations thereof.
 8. The method of claim 1,wherein said AEX column comprises an anionic exchange substituentincluding diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) andquaternary amine (Q) groups.
 9. The method of claim 1, furthercomprising after binding aflibercept from said clarified harvest,subjecting said aflibercept to one or more further chromatographic stepsselected from the group consisting of: cation exchange chromatography,hydrophobic interactive chromatography, size exclusion chromatographyand a combination thereof.
 10. The method of claim 1, wherein saideluate comprises acidic species of aflibercept and wherein said acidicspecies of aflibercept correspond to peaks that elute earlier than amain peak in a cation exchange chromatography (CEX) chromatogram ofaflibercept, and wherein said chromatogram is generated using a firstmobile phase of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7and a second mobile phase of 40 mM sodium phosphate, 100 mM sodiumchloride pH 9.0 (Mobile phase B), and wherein said chromatogram isgenerated using detection at 280 nm.
 11. The method of claim 10, whereinsaid percent of acidic species of aflibercept in said affinity eluate isgreater than said percent of acidic species of aflibercept in said oneor more flowthrough fractions when said concentrations of protein insaid eluate and flowthrough fractions are normalized.
 12. The method ofclaim 11, wherein said acidic species of aflibercept in said affinityeluate are reduced by at least ten percent compared to said flowthroughfraction when said concentrations of said affinity eluate andflowthrough protein are normalized.
 13. The method of claim 11, whereinsaid aflibercept from said one or more flowthrough fractions comprisesless than 20% total acidic species of aflibercept.
 14. The method ofclaim 11, wherein said acidic species of aflibercept comprisesaflibercept having at least one oxidized amino acid residue selectedfrom group consisting of methionine, tryptophan, histidine,phenylalanine, tyrosine and a combination thereof.
 15. The method ofclaim 1, wherein said AEX column is equilibrated using an equilibrationbuffer and washed using a wash buffer and wherein pH of both saidequilibration and wash buffers is about 8.30 to about 8.60.
 16. Themethod of claims 1, wherein said AEX column is equilibrated using anequilibration buffer and washed using a wash buffer and wherein saidconductivity of both said equilibration and wash buffers is about 1.50to about 3.0 mS/cm.
 17. The method of claim 1, wherein said eluatecomprises oxidized species of aflibercept and wherein said oxidizedspecies of aflibercept is measured by subjecting said affinity eluateand said flowthrough fractions to digestion, followed by their analysisusing reverse-phase ultra-performance chromatography (UPLC), detectionat wavelengths of 280 nm, 320 nm and 350 nm and mass spectrometryanalysis using said first mobile phase of 0.1% formic acid in water anda second mobile phase of was 0.1% formic acid in acetonitrile.
 18. Themethod of claim 17, wherein said percent of oxidized species ofaflibercept in said affinity eluate is greater than said percent ofoxidized species of aflibercept in said flowthrough fraction.
 19. Themethod of claim 18, wherein said percent of oxidized species ofaflibercept in said flowthrough fraction are reduced by at least about10% compared to said percent of oxidized species of aflibercept in saidaffinity eluate.
 20. The method of claim 18, wherein said oxidized aminoacid residue is selected from the group consisting of methionine,tryptophan, histidine, phenylalanine, tyrosine and a combinationthereof.
 21. The method of claim 18, wherein said oxidized species ofaflibercept has an amino acid residue which is selected from an aminoacid residue on a polypeptide having an amino acid sequence as set forthin the group consisting of: SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.:19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, andSEQ ID NO.:
 67. 22. The method of claim 18, wherein said protein furthercomprises one or more variant amino acid residues selected from apolypeptide having an amino acid sequence as set forth in the groupconsisting of: SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ IDNO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, SEQ ID NO.: 56,SEQ ID NO.: 64, SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ ID NO.: 67, SEQ IDNO.: 68, SEQ ID NO.: 69, SEQ ID NO.: 70, SEQ ID NO.: 71, andcombinations thereof.
 23. The method of claim 1, wherein saidpolypeptide capable of binding to or interacting with said afliberceptcomprises an isolated amino acid sequence selected from a SEQ ID NO.:72, SEQ ID NO.: 73, SEQ ID NO.: 74, SEQ ID NO.: 75, SEQ ID NO.: 76, SEQID NO.: 77, SEQ ID NO.: 78, SEQ ID NO.: 79 and SEQ ID NO.:
 80. 24. Themethod of claim 1, further comprising equilibrating said affinitychromatography column of (b) using an equilibration buffer.
 25. Themethod of claim 24, wherein said equilibration buffer is Dulbecco'sPhosphate-Buffered Saline or Tris hydrochloride.
 26. The method of claim25, wherein said equilibration buffer has a pH of about 8.3 to about8.6.
 27. The method of claim 1, further comprising washing column (d)with an equilibration buffer to obtain one or more flowthroughfractions.
 28. The method of claim 1, further comprising subjectingcolumn of (b) with an elution buffer to obtain one or more elutedfractions.
 29. The method of claim 28, further comprising neutralizingsaid eluted fractions with said addition of a neutralizing buffer. 30.The method of claim 1, wherein an amount of host-cell proteins in (c) issignificantly reduced by about 90%, about 95%, about 98%, or about 99%as compared to said amount of host-cell proteins in said clarifiedharvest.